Composition comprising amino acid polymers and a bioactive agent and method of preparing thereof

ABSTRACT

A method of treating, reducing or preventing bacterial infection in a wound, the method comprising: applying a film on the wound, the film including a biodegradable polymer with bacteriophages dispersed therein, wherein the polymer is a poly (ester amide urea).

FIELD OF THE INVENTION

The present invention relates to compositions comprising amino acidpolymers and a bioactive agent and method of preparing thereof. Also,the present invention relates to the field of wound treatment, and ismore particularly concerned with a method for treating a wound with abacteriophages containing thin films.

BACKGROUND

Millions of people worldwide suffer abrasions and acute and chronicwounds each year. Wound dressings are designed with care so that theirapplication will not infect or inflame a wound. Additionally, many wounddressings are designed to provide therapeutic benefits. For example,wound dressings comprising a polymer and bactericidal substances arecommonly used in the treatment of superficial wounds. Additional wounddressings utilized for wound healing may further include at least oneadditional bioactive agent (e.g., pain relievers or bactericidalsubstances) released in a controlled manner as a result of diffusion,enzymatic degradation (e.g., proteolytic degradation), surface erosion,bulk erosion, or combinations thereof.

Many wound dressings comprising a polymer and at least one bioactiveagent can result in positive clinical outcomes when used to treatsuperficial wounds. However, management of deep wounds in poorlyvascularized tissues (e.g., trophic ulcers and bedsores) remainschallenging for many patients. As used herein, “patient” refers tohumans and other animals (e.g., mammals).

Deep wound infections are often difficult to treat and frequently becomeinfected by multiple pathogenic organisms due to insufficient immuneresponse in areas with minimal vasculature. Antibiotics may penetratepoorly into deep wounds, making eradication of infection extremelydifficult.

While wound dressings comprising a polymer and at least one antibioticmay be used in the treatment of both superficial wounds and deep wounds,their efficacy may be increasingly limited by the development ofantibiotic resistance at the wound site. Recently, there has beeninterest in using bactericidal substances such as silver sulfadiazine(and related diazine derivatives of sulfanilamide), furagin (and/orpharmaceutically acceptable salts thereof) and chlorhexidine (and/orpharmaceutically acceptable salts thereof) in addition to or in place ofantibiotics in antibacterial wound dressings. However, utilization ofsuch substances may be limited by their inherent toxicity, particularlyin patients with underlying kidney or liver disease.

Bactericidal substances of natural origin, including highly specificviruses that can infect bacteria, referred to herein as bacteriophages,may present a promising alternative treatment. Bacteriophages are alsoreferred to as “phages” herein. Bacteriophages have been reported to beeffective in treating skin infections caused by Pseudomonas bacteria,Staphylococcus bacteria, Kiebsiella bacteria, Proteus bacteria,Escherichia coli, and other pathogenic bacterial species. Bacteriophagetends to be highly specific for certain bacteria, so bacteriophagetherapy may be targeted to kill specific pathogens without disturbingnormal bacterial flora.

Polymers mixed with bacteriophage may be superior to liquid preparationsof bacteriophage for the treatment of deep and chronic wounds due to thepotential for controlled release of the bacteriophage. However, manycurrently available polymer-bacteriophage compositions compriseimmobilized bacteriophage with reduced bactericidal activity or polymersthat are not biodegradable, necessitating deliberate removal of thewound dressing. Thus, there remains a need in the art for polymers thatcan be prepared under mild conditions without using toxic catalysts andthat can degrade by erosion into neutral byproducts, such as normalproducts of human metabolism. In some embodiments, the aforementionedpolymers may be useful in the treatment of both superficial and deepwounds.

SUMMARY OF THE INVENTION

In a broad aspect, there is provided a method of treating, reducing orpreventing bacterial infection in a wound, the method comprising:applying a flexible film on the wound, the film including abiodegradable polymer with bacteriophages dispersed therein, wherein thepolymer is a poly (ester amide urea) comprising the following two blockswith random distribution thereof:

wherein

the ratio of l:m ranges from 0.01:0.99 to 0.99:0.01, l+m=1,

R₁ is chosen from C₁-C₁₂ alkylene optionally interrupted by at least oneoxygen, C₃-C₈ cycloalkylene, C₃-C₁₀ cycloalkylalkylene,

R₃ is C₁-C₁₂ alkylene,R₂ and R₄ are independently chosen from the side chains of L- andD-amino acids so that the carbon to which R₂ or R₄ is attached has L orD chirality.

Typically, the film is a dry solid film.

There may also be provided a method wherein R₁ is —(CH₂)₆—, R₃ is—(CH₂)₈—, and both R₂ and R₄ are the side chain of L-leucine.

There may also be provided a method wherein the film further includessalt particles dispersed in the polymer.

There may also be provided a method wherein at least part of thebacteriophages is adsorbed on the salt particles.

There may also be provided a method wherein the salt particles includeCaCO₃ particles.

There may also be provided a method wherein the film further includes abuffer.

There may also be provided a method wherein the buffer is TMN(Tris-MgCl₂—NaCl) buffer.

There may also be provided a method wherein the film further includes atleast one of silversulfadiazine, silver nitrate and nanocrystallinesilver.

There may also be provided a method wherein the film further includes anantibiotic.

There may also be provided a method wherein the film is applied for aduration of between 1 day and 20 days.

There may also be provided a method wherein the film is applied for aduration of between 3 and 7 days.

There may also be provided a method further comprising removing the filmafter the duration and applying another similar film on the wound.

There may also be provided a method further comprising delivering apredetermined fraction of the bacteriophages in the wound within apredetermined rapid delivery period immediately after applying the film,the predetermined rapid delivery period being smaller than 4 hours, andafterwards releasing at least part of the remaining bacteriophages inthe wound at a rate smaller than within the predetermined rapid deliveryperiod.

There may also be provided a method further comprising graduallydelivering at least part of the bacteriophages in the wound at acontrolled delivery rate, the controlled delivery rate being controlledby a volume and composition of a wound exudate produced by the woundwhile the film is applied.

There may also be provided a method wherein the controlled delivery rateincreases for an increase is volumic production rate of the woundexudate.

There may also be provided a method wherein the controlled delivery rateincreases with an increase in elastase and metalloproteinases quantitypresent in the wound exudate as a result of cell lysis within the wound.

There may also be provided a method wherein the film includes an enzymeoperative to degrade the film.

There may also be provided a method wherein the enzyme is selected fromelastase and a metalloproteinase.

There may also be provided a method wherein the bacteriophages arepresent in the film at 500,000 PFU/cm{circumflex over ( )}2 or more.

There may also be provided a method wherein the film is non-woven andnon-porous.

There may also be provided a method wherein the film is between 100 μmand 1000 μm thick.

There may also be provided a method wherein the wound is a pressureulcer.

There may also be provided a method wherein the wound is a burn wound.

There may also be provided a method wherein a bactericide is applied tothe wound before the film is applied.

There may also be provided a method wherein the bactericide includes atleast one of an antibiotic, silversulfadiazine, silver nitrate andnanocrystalline silver.

There may also be provided a method wherein the wound contains at leastone of antibiotic-resistant and silver-resistant bacteria, thebacteriophages being specific for the at least one ofantibiotic-resistant and silver-resistant bacteria.

There may also be provided a method wherein the film is substantiallytransparent when wet.

In another broad aspect, there is provided a method of treating,reducing or preventing bacterial infection in a wound, the methodcomprising: applying a film on the wound, the film includingbacteriophages dispersed in a biodegradable polymer, wherein the polymeris selected from

(1) a poly (ester amide urea) wherein at least one diol, at least onediacid, and at least one amino acid are linked together through an esterbond, an amide bond, and a urea bond,

(2) a poly (ester urethane urea) wherein at least one diol and at leastone amino acid are linked together through an ester bond, a urethanebond, and a urea bond,

(3) a poly (ester amide urethane urea) wherein at least one diol, atleast one diacid, and at least one amino acid are linked togetherthrough an ester bond, an amide bond, a urethane bond, and a urea bond,

(4) a poly (ester amide urethane) wherein at least one diol, at leastone diacid, and at least one amino acid are linked together through anester bond, an amide bond, and a urethane bond,

(5) a poly (ester urea) wherein at least one diol and at least one aminoacid are linked together through an ester bond and a urea bond, and

(6) a poly (ester urethane) wherein at least one diol and at least oneamino acid are linked together through an ester bond and a urethanebond,

further wherein

the at least one diol is a compound of formula:

HO—R₁—OH, R₁ is chosen from C₁-C₁₂ alkylene optionally interrupted by atleast one oxygen, C₃-C₈ cycloalkylene, C₃-C₁₀ cycloalkylalkylene,

the at least one diacid is a compound of formula:HO—(CO)—R₃—(CO)—OH, R₃ is C₁-C₁₂ alkylene,the at least one amino acid is chosen from a naturally occurring aminoacid and non-naturally occurring amino acid.

The various additional features mentioned in the first method providedabove also apply to the present method.

Advantageously, in some embodiments, the proposed film produces oreliminates toxic organic by-products during the manufacturing process,for example by using triphosgene as one of the base reactants, whichalso advantageously provides a bacteriophage friendly environment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-C depicts the UV spectra of Staphylophage, MgCO₃/Staphylophage,F₀ obtained after step 2 (Example 4), and MgCO₃/Staphylophage, F1obtained after step 3 (Example 4), respectively.

FIG. 2A-C depicts the UV spectra of Staphylophage, CaCO₃/Staphylophage,F₀ obtained after step 2 (Example 4), and CaCO₃/Staphylophage, F1obtained after step 3 (Example 4), respectively.

FIG. 3 depicts the appearance of an exemplary perforated polymeric film.

FIG. 4 shows a representative example of the antibacterial activity ofStaphylophage adsorbed on CaCO₃, the wet solid after Step 1 and 2(Example 4), in a double agar overlay assay.

FIG. 5 shows representative examples of the antibacterial activity ofIntestiphage adsorbed on CaCO₃, the wet solid after Step 1 and 2(Example 4), in a double agar overlay assay.

FIG. 6A-D compares the effect on bacterial growth of bacteriophages andSilverlon™ taken alone or in combination on 4 bacterial strains.

FIG. 7 illustrates the effect of a proposed film including bacteriophageon bacterial growth in wound analogs in vitro.

DETAILED DESCRIPTION

Disclosed herein are: (1) a polymer that can be biodegradable; (2) acomposition comprising the polymer, a bioactive agent, and optionally afiller; and (3) methods of preparation thereof. Also disclosed herein isa composition in a powdery form comprising a bacteriophage.

Definitions and Abbreviations

A dash (“-”) that is not between two letters or symbols is used toindicate a point of attachment for a substituent. For example, —(CH₂)₆—is attached on each end through a carbon atom.

Unless clearly indicated otherwise, use of the terms “a,” “an,” and thelike refers to one or more.

The term “alkylene” herein refers to a bivalent hydrocarbon groupselected from linear and branched saturated hydrocarbon groupscomprising, for example, from 1 to 18 carbon atoms, such as from 1 to12, further such as from 1 to 10, even further such as from 1 to 6,carbon atoms. Non-limiting examples of alkylene include —CH₂—, —(CH₂)₂—,—(CH₂)₆—, and —(CH₂)₈—. When “alkylene” is interrupted by at least oneoxygen, it means that at least one pair of neighboring carbons in analkylene is separated by one oxygen, such as in the ether groups—CH₂—O—CH₂—, —CH₂—CH₂—O—CH₂—CH₂—, and —CH₂—O—CH₂—CH₂—O—CH₂—CH₂—.

The term “cycloalkylene” herein refers to a bivalent hydrocarbon groupselected from saturated cyclic hydrocarbon groups, comprising monocyclicand polycyclic (e.g., bicyclic and tricyclic) groups, wherein the twopoints of attachment are on the cyclic ring. For example, the cycloalkylgroup may comprise from 3 to 12 carbon atoms, such as from 3 to 8,further such as from 3 to 6, from 3 to 5, or from 3 to 4, carbon atoms.Non-limiting examples of cycloalkylene include

The term “cycloalkylalkylene” herein refers to a bivalent hydrocarbongroup wherein a saturated cyclic hydrocarbon group, chosen frommonocyclic and polycyclic (e.g., bicyclic and tricyclic) groups, issubstituted by at least one hydrocarbon group chosen from linear andbranched saturated hydrocarbon group comprising, for example, from 1 to18 carbon atoms, and wherein at least one of the two points ofattachment is on the at least one hydrocarbon group chosen from linearand branched saturated hydrocarbon groups. Non-limiting examples ofcycloalkylalkylene include

As used herein, the terms “L-amino acid” and “D-amino acid” (orD-enantiomer) refers to amino acid stereoisomers. All known naturallyoccurring amino acids except for glycine, which adopts a singleconformation, can exist in two isomeric states. L-amino acids andD-amino acids are analogous to left-handed and right-handed enantiomers.L-amino acids are primarily utilized in mammalian cells to produceproteins.

As used herein, a “patient” refers to a human or other animal, forexample a mammal. In some embodiments, the patient is a mammal, and inother specific embodiments, the patient is human.

As used herein, a compound that is “poorly soluble” in a solvent is acompound that is “slightly soluble,” “very slightly soluble,” or“practically insoluble” in the solvent according to the definitionsprovided in the United States Pharmacopeia (USP). The USPclassifications for solubility are listed in Supplemental Table 1 below.

Supplemental Table 1. USP Definitions of Solubility Pads of solventSolubility Solubility Description form required for one part rangeassigned (Solubility definition) of solute (mg/mL) (mg/mL) Very soluble(VS) <1 >1,000 1,000 Freely soluble (FS) from 1 to 10   100-1,000 100Soluble from 10 to 30  33-100 33 Sparingly soluble (SPS) from 30 to 10010-33 10 Slightly soluble (SS) from 100 to 1,000  1-10 1 Very slightlysoluble (VSS) from 1,000 to 10,000 0.1-1   0.1 Practically insoluble(PI) >10,000 <0.1 0.01

As used herein, a “polymer blend” is a mixture comprising at least twopolymers, wherein the two polymers are not the same.

Polymers and Polymer Blends

In some embodiments, the disclosure provides an amino acid based polymerthat can be biodegradable.

Amino acid based polymers that can be biodegradable are suitable forpreparing a composition further comprising at least one bioactive agent,such as bacteriophage. For example, in some embodiments, bacteriophageis dispersed, mixed, dissolved, homogenized, or covalently bonded in acomposition further comprising an amino acid based polymer.

The amino acid based polymers can be solubilized in chloroform, anorganic solvent which may not inactivate the at least one bioactiveagent such as bacteriophage during the preparation of the composition.

In some embodiments, the polymer can be solubilized in an organicsolvent. For example, in some embodiments, the polymer can besolubilized in chloroform, dichloromethane, or ethyl acetate.

Other biodegradable polymers, including commercially availablepoly(lactide/glycolide) copolymers, are also soluble in chloroform.However, these biodegradable polymers can be used but are less suitablefor preparing a composition further comprising at least one bioactiveagent, such as the at least one bioactive agent disclosed herein, sincethey may have some limitations compared to amino acid basedbiodegradable polymers, which are listed in Table 1 below:

TABLE 1 Amino acid based biodegradable polymers vs.poly(lactide/glycolide) copolymers Amino acid based biodegradablepolymers (Properties achieved in Poly(lactide/glycolide) copolymers someembodiments) (General properties) Polycondensation synthesis withoutRing-opening polymerization using toxic metalorganic using any toxiccatalyst catalysts Synthesis under normal atmospheric Synthesis viaring-opening polymerization under conditions at room temperatureextra-dry conditions at 195-230° C. Higher hydrophilicity and, hence,better Hydrophobic polymers, relatively poor compatibility compatibilitywith tissues with tissues Longer shelf-life Thermodynamically unstablepolymers, depolymerize on storage A wide range of material propertiesNarrow range of desirable material properties, poor including elasticitydesirable for wound elasticity dressings A high range of biodegradationrates A narrow range of biodegradation rates, poor possibility that canbe regulated by impregnating to regulate them by impregnating enzymesenzymes A variable hydrophobicity Low variability ofhydrophobicity/hydrophilicity hydrophilicity balance suitable forbalance suitable for constructing a drug sustained/ constructing a drugsustained/ controlled release devices controlled release devices Highnutritious properties owing to Poor nutritious properties (no amino acidreleased upon release of α-amino acids upon biodegradation)biodegradation Lower acidic or neutral medium after Higher acidic mediumafter ultimate biodegradation¹ ultimate biodegradation*

Notes on the * in the table: After ultimate biodegradation ofpoly(glycolic acid), poly(lactic acid)s, or poly(lactic-co-glycolicacid), glycolic and lactic acids are released that have pK_(a) 3.83 and3.86, accordingly. After ultimate biodegradation of poly(ester amide)s,e.g. on the basis of sebacic acid, neutral α-amino acids (zwitterioniccompounds) and fatty diols along with sebacic acid are released; thelatter has pK_(a1) 4.72 and pK_(a2) 5.45 that is by approximately oneunit of pK_(a) lower when compared to glycolic and lactic acids. Afterultimate biodegradation of amino acid based poly(ester urea)s, themedium should be close to physiological since normal products ofmetabolism—carbon dioxide and α-amino acids along with neutral fattydiols are released. The preparation of poly(ester amide)s is describedin R. Katsarava, V. Beridze, N. Arabuli, D. Kharadze, C. C. Chu, C. Y.Won. Amino acid based bioanalogous polymers. Synthesis and study ofregular poly(ester amide)s based on bis(α-amino acid) α,ω-alkylenediesters and aliphatic dicarboxylic acids. J. Polym. Sci.: Part A:Polym. Chem. 37, 391-407 (1999)

In addition, biodegradation of poly(glycolic acid) and poly(lacticacid)s may lead to the production of much higher quantities of acidicproducts per unit weight of the polymers are released compared topoly(ester amide)s. For example:

-   -   after biodegradation of 1.0 g of poly(glycolic acid), 1.31 g        (0.017 mole) of glycolic acid is released,    -   after biodegradation of 1.0 g of poly(lactic acid), 1.25 g        (0.014 mole) of lactic acid is released,    -   after biodegradation of 1.0 g of poly(ester amide) composed of        phenylalanine, sebacic acid and 1,6-hexanediol, only 0.35 g        (0.0017 mole) of sebacic acid is released, and    -   after biodegradation of 1.0 g of co-poly(ester urea amide),        which can be useful, for example, for preparing bacteriophage        containing composition, a negligible quantity—0.09 g (0.00044        mole) of sebacic acid is released.    -   after biodegradation of co-poly(ester urea urethane), normal        products of metabolism—carbon dioxide and α-amino acids along        with neutral fatty diols are released (i.e., no acidic component        is released).

A highly acidic medium may be harmful to some bioactive agents such asbacteriophage. Therefore, poly(lactide/glycolide) polyesters may be lesspromising than amino acid based polymers that can be bio-degradable forpreparing a composition further comprising at least one bioactive agentsuch as bacteriophage.

In addition, polymers provided herein may be synthesized using organicsolvents that are compatible with bacteriophage. For example, polymersprovided herein may be synthesized using organic solvents such aschloroform, dichloromethane, and ethyl acetate as opposed todimethylformamide (DMF), dimethylacetamide (DMA), and dimethylsulfoxide(DMSO). The organic solvents DMF, DMA, and DMSO, which have previouslybeen used in the synthesis of bio-degradable amino acid based polymers,are not compatible with bacteriophage, rendering polymers produced usingthese solvents less suitable for the preparation of compositionscomprising bacteriophage.

In addition, in some embodiments, polymers provided herein do notcomprise L-phenylalanine, an amino acid found in some previous polymericblends used for the preparation of compositions further comprising atleast one bioactive agent. The presence of L-phenylalanine in a polymermay led to adverse events in patients suffering from phenylketonuria.

Exemplary amino acids used to produce polymers described herein include,but are not limited to, L-glycine, L-alanine, L-valine, L-leucine,L-isoleucine, L-proline, L-methionine, L-phenylalanine, L-tryptophan,and D isomers thereof.

Provided herein is a polymer chosen from

(1) a poly (ester amide urea) wherein at least one diol, at least onediacid, and at least one amino acid are linked together through an esterbond, an amide bond, and a urea bond,

(2) a poly (ester urethane urea) wherein at least one diol and at leastone amino acid are linked together through an ester bond, a urethanebond, and a urea bond,

(3) a poly (ester amide urethane urea) wherein at least one diol, atleast one diacid, and at least one amino acid are linked togetherthrough an ester bond, an amide bond, a urethane bond, and a urea bond,

(4) a poly (ester amide urethane) wherein at least one diol, at leastone diacid, and at least one amino acid are linked together through anester bond, an amide bond, and a urethane bond,

(5) a poly (ester urea) wherein at least one diol and at least one aminoacid are linked together through an ester bond and a urea bond (in otherwords, at least one diol, a carbonic acid, and at least one amino acidare linked together through an ester bond and a urea bond), and

(6) a poly (ester urethane) wherein at least one diol and at least oneamino acid are linked together through an ester bond and a urethanebond,

further wherein

the at least one diol is a compound of formula HO—R₁—OH, wherein R₁ ischosen from an alkylene optionally interrupted by at least one oxygen, acycloalkylene, a cycloalkylalkylene,

the at least one diacid is a compound of formula HO—(CO)—R₃—(CO)—OH,wherein R₃ is an alkylene, and

the at least one amino acid is chosen from a naturally occurring aminoacid or a non-naturally occurring amino acid.

Further provided herein is a polymer chosen from

(1) a poly (ester amide urea) wherein at least one diol, at least onediacid, and at least one amino acid are linked together through an esterbond, an amide bond, and a urea bond;

(2) a poly (ester urethane urea) wherein at least one diol and at leastone amino acid are linked together through an ester bond, a urethanebond, and a urea bond;

(3) a poly (ester amide urethane urea) wherein at least one diol, atleast one diacid, and at least one amino acid are linked togetherthrough an ester bond, an amide bond, a urethane bond, and a urea bond;

(4) a poly (ester amide urethane) wherein at least one diol, at leastone diacid, and at least one amino acid are linked together through anester bond, an amide bond, and a urethane bond;

(5) a poly (ester urea) wherein at least one diol and at least one aminoacid are linked together through an ester bond and a urea bond (in otherwords, at least one diol, a carbonic acid, and at least one amino acidare linked together through an ester bond and a urea bond); and

(6) a poly (ester urethane) wherein at least one diol and at least oneamino acid are linked together through an ester bond and a urethanebond,

further wherein

the at least one diol is a compound of formula HO—R₁—OH, wherein R₁ ischosen from C₁-C₁₂ alkylene optionally interrupted by at least oneoxygen, C₃-C₈ cycloalkylene, C₃-C₁₀ cycloalkylalkylene,

the at least one diacid is a compound of formula HO—(CO)—R₃—(CO)—OH,wherein R₃ is chosen from C₁-C₁₂ alkylene; and

the at least one amino acid is a naturally occurring amino acid or anon-naturally occurring amino acid, such as an L-amino acid or D-aminoacid, further such as L-glycine, L-alanine, L-valine, L-leucine,L-isoleucine, L-proline, L-methionine, L-phenylalanine, or L-tryptophan,or D isomers thereof.

In some embodiments, R₁ is chosen from C₂-C₁₂ alkylene optionallyinterrupted by at least one oxygen, C₃-C₈ cycloalkylene, C₃-C₁₀cycloalkylalkylene,

In some embodiments, R₃ is chosen from C₂-C₁₂ alkylene.

In some embodiments, the polymer is selected from

(1) a poly (ester amide urea) wherein at least one diol, at least onediacid, and at least one amino acid are linked together through an esterbond, an amide bond, and a urea bond;

(2) a poly (ester urethane urea) wherein at least one diol and at leastone amino acid are linked together through an ester bond, a urethanebond, and a urea bond;

(3) a poly (ester amide urethane urea) wherein at least one diol, atleast one diacid, and at least one amino acid are linked togetherthrough an ester bond, an amide bond, a urethane bond, and a urea bond;and

(4) a poly (ester amide urethane) wherein at least one diol, at leastone diacid, and at least one amino acid are linked together through anester bond, an amide bond, and a urethane bond,

further wherein

the at least one diol is a compound of formula HO—R₁—OH, wherein R₁ ischosen from C₁-C₁₂ alkylene optionally interrupted by at least oneoxygen, C₃-C₈ cycloalkylene, C₃-C₁₀ cycloalkylalkylene,

the at least one diacid is a compound of formula HO—(CO)—R₃—(CO)—OH,wherein R₃ is C₁-C₁₂ alkylene; and the at least one amino acid is anaturally occurring amino acid or non-naturally occurring amino acid,such as L-amino acid or D-amino acid, further such as L-valine,L-leucine, L-isoleucine, L-methionine, or L-phenylalanine, or D isomersthereof.

In some embodiments, R₁ is chosen from C₂-C₁₂ alkylene optionallyinterrupted by at least one oxygen, C₃-C₈ cycloalkylene, C₃-C₁₀cycloalkylalkylene,

In some embodiments, R₃ is chosen from C₂-C₂ alkylene.

In some embodiments, the polymer is grindable. In some embodiments, thegrindable polymer is poly (ester urea). In some embodiments, thegrindable polymer is poly (ester urea) with low molecular weight, suchas 5-6 kDa weight average.

In some embodiments, the polymer is the poly (ester amide urea). In someembodiments, the poly (ester amide urea) comprises the following twoblocks with random distribution thereof:

wherein

the range of l:m ratio is from 0.01:0.99 to 0.99:0.01, l+m=1, such as0.05:0.95 to 0.95:0.05, and further such as 0.10:0.90 to 0.90:0.10;

R₁ is chosen from C₁-C₁₂ alkylene optionally interrupted by at least oneoxygen, C₃-C₈ cycloalkylene, C₁-C₁₀ cycloalkylalkylene,

R₃ is chosen from C₁-C₁₂ alkylene; and

R₂ and R₄ are independently chosen from the side chains of L- andD-amino acids such that the carbon to which R₂ or R₄ is attached has Lor D chirality. For example, R₂ and R₄ are independently chosen from—CH(CH₃)₂, —CH₂CH(CH₃)₂, —CH(CH₃)CH₂CH₃, and —CH₂C₆H₅. R₂ and R₄ canalso be independently chosen from —(CH₂)₃CH₃ and —(CH₂)₃SCH₃, such thatthe carbons to which they are attached have R or S chirality.

In some embodiments, R₁ is chosen from C₂-C₁₂ alkylene optionallyinterrupted by at least one oxygen, C₃-C₆ cycloalkylene, C₃-C₁₀cycloalkylalkylene,

In some embodiments, R₃ is chosen from C₂-C₁₂ alkylene.

In some embodiments of the poly (ester amide urea), R₁ is —(CH₂)₆—. Insome embodiments of the poly(ester amide urea), R₃ is —(CH₂)₆—. In someembodiments of the poly (ester amide urea), R₂ and R₄ are chosen fromthe side chain of L-leucine.

In some embodiments, the polymer is the poly (ester urethane urea). Insome embodiments, the poly (ester urethane urea) comprises the followingtwo blocks with random distribution thereof:

wherein

the ratio of l:m ranges from 0.01:0.99 to 0.99:0.01, l+m=1, such as0.05:0.95 to 0.95:0.05, and further such as 0.10:0.90 to 0.90:0.10;

R₁ and R₅ are independently chosen from C₁-C₁₂ alkylene optionallyinterrupted by at least one oxygen, C₃-C₈ cycloalkylene, C₃-C₁₀cycloalkylalkylene,

and

R₂ and R₄ are independently chosen from the side chains of L- andD-amino acids such that the carbon to which R₂ or R₄ is attached has Lor D chirality. For example, R₂ and R₄ are independently chosen from—CH(CH₃)₂, —CH₂CH(CH₃)₂, —CH(CH₃)CH₂CH₃, and —CH₂C₆H₅. R₂ and R₄ canalso be independently chosen from —(CH₂)₃CH₃ and —(CH₂)₃SCH₃, with thecarbons to which they are attached having R or S chirality.

In some embodiments, R₁ and R₅ are independently chosen from C₂-C₁₂alkylene optionally interrupted by at least one oxygen, C₃-C₈cycloalkylene, C₃-C₁₀cycloalkylalkylene,

In some embodiments of the poly (ester urethane urea), R₁ is —(CH₂)₆—.In some embodiments of the poly (ester urethane urea), R₃ is —(CH₂)₈—.In some embodiments of the poly (ester urethane urea), R₂ and R₄ arechosen from the side chain of L-leucine.

In some embodiments, the polymer is the poly (ester amide urethaneurea). In some embodiments, the poly (ester amide urethane urea)comprises the following three blocks with random distribution thereof:

wherein

the ratio of l:m:k ranges from 0.05:0.05:0.90 to 0.90:0.05:0.05,l+m+k=1;

R₁ and R₅ are independently chosen from C₁-C₁₂ alkylene optionallyinterrupted by at least one oxygen, C₃-C₈ cycloalkylene, C₃-C₁₀cycloalkylalkylene,

R₃ is C₁-C₁₂ alkylene; and

R₂ and R₄ are independently chosen from the side chains of L- andD-amino acids such that the carbon to which R₂ or R₄ is attached has Lor D chirality. For example, R₂ and R₄ are independently chosen from—CH(CH₃)₂, —CH₂CH(CH₃)₂, —CH(CH₃)CH₂CH₃, and —CH₂C₆H₅; R₂ and R₄ areindependently chosen from —(CH₂)₃CH₃ and —(CH₂)₃SCH₃, with the carbonsto which they are attached having R or S chirality.

In some embodiments, R₁ and R₅ are independently chosen from C₂-C₁₂alkylene optionally interrupted by at least one oxygen, C₃-C₈cycloalkylene, C₃-C₁₀ cycloalkylalkylene,

In some embodiments, R₃ is chosen from C₂-C₁₂ alkylene.

In some embodiments of the poly (ester amide urethane urea), R₁ is—(CH₂)₆—. In some embodiments of the poly (ester amide urethane urea),R₃ is —(CH₂)₈—. In some embodiments of the poly (ester amide urethaneurea), R₂ and R₄ are chosen from the side chain of L-leucine.

In some embodiments, the polymer is the poly (ester amide urethane).

In some embodiments, the poly (ester amide urethane) comprises thefollowing two blocks with random distribution thereof:

wherein

the ratio of l:m ranges from 0.01:0.99 to 0.99:0.01, l+m=1, such as0.05:0.95 to 0.95:0.05, and further such as 0.10:0.90 to 0.90:0.10;

R₁ and R₅ are independently chosen from C₁-C₁₂ alkylene optionallyinterrupted by at least one oxygen, C₃-C₈ cycloalkylene, C₃-C₁₀cycloalkylalkylene,

R₃ is C₁-C₁₂ alkylene; and

R₂ and R₄ are independently chosen from the side chains of L- andD-amino acids such that the carbon to which R₂ or R₄ is attached has Lor D chirality. For example, R₂ and R₄ are independently chosen from—CH(CH₃)₂, CH₂CH(CH₃)₂, CH(CH₃)CH₂CH₃, and CH₂C₆H₅; R₂ and R₄ can alsobe independently chosen from (CH₂)₃CH₃ and (CH₂)₃SCH₃, with the carbonsto which they are attached having R or S chirality.

In some embodiments, R₁ and R₅ are independently chosen from C₂-C₁₂alkylene optionally interrupted by at least one oxygen, C₃-C₈cycloalkylene, C₃-C₁₀ cycloalkylalkylene,

In some embodiments, R₃ is chosen from C₂-C₁₂ alkylene.

In some embodiments of the poly (ester amide urethane), R₁ is —(CH₂)₆—.

In some embodiments of the poly (ester amide urethane), R₃ is —(CH₂)₈—.

In some embodiments of the poly (ester amide urethane), R₂ and R₄ arechosen from the side chain of L-leucine.

Also provided herein is a polymer blend comprising a first polymer and asecond polymer, wherein the first polymer is chosen from:

(1) a poly (ester amide urea) wherein at least one diol, at least onediacid, and at least one amino acid are linked together through an esterbond, an amide bond, and a urea bond;

(2) a poly (ester urethane urea) wherein at least one diol and at leastone amino acid are linked together through an ester bond, a urethanebond, and a urea bond;

(3) a poly (ester amide urethane urea) wherein at least one diol, atleast one diacid, and at least one amino acid are linked togetherthrough an ester bond, an amide bond, a urethane bond, and a urea bond;

(4) a poly (ester amide urethane) wherein at least one diol, at leastone diacid, and at least one amino acid are linked together through anester bond, an amide bond, and a urethane bond;

(5) a poly (ester urea) wherein at least one diol and at least one aminoacid are linked together through an ester bond and a urea bond (in otherwords, at least one diol, a carbonic acid, and at least one amino acidare linked together through an ester bond and a urea bond); and

(6) a poly (ester urethane) wherein at least one diol and at least oneamino acid are linked together through an ester bond and a urethanebond,

further wherein

the at least one diol is a compound of formula HO—R₁—OH, wherein R₁ ischosen from C₁-C₁₂ alkylene optionally interrupted by at least oneoxygen, C₃-C₈ cycloalkylene, C₃-C₁₀ cycloalkylalkylene,

the at least one diacid is a compound of formula HO—(CO)—R₃—(CO)—OH,wherein R₃ is chosen from C₁-C₁₂ alkylene; and

the at least one amino acid is a naturally occurring amino acid or anon-naturally occurring amino acid, such as an L-amino acid or D-aminoacid, further such as L-glycine, L-alanine, L-valine, L-leucine,L-isoleucine, L-proline, L-methionine, L-phenylalanine, or L-tryptophan,or D isomers thereof; and

the second polymer is a poly (ester amide) wherein at least one diol, atleast one diacid and at least one amino acid are linked together throughan ester bond and an amide bond,

wherein the at least one diol is a compound of formula HO—R₁—OH, whereinR₁ is chosen from C₁-C₁₂ alkylene optionally interrupted by at least oneoxygen, C₃-C₈ cycloalkylene, C₃-C₁₀ cycloalkylalkylene,

the at least one diacid is a compound of formula HO—(CO)—R₃—(CO)—OH,wherein R₃ is chosen from C₁₋₂ alkylene; and

the at least one amino acid is a naturally occurring amino acid or anon-naturally occurring amino acid, such as an L-amino acid or D-aminoacid, further such as L-glycine, L-alanine, L-valine, L-leucine,L-isoleucine, L-proline, L-methionine, L-phenylalanine, or L-tryptophan,or D isomers thereof, or the second polymer is a polymer chosen from

(1) a poly (ester amide urea) wherein at least one diol, at least onediacid, and at least one amino acid are linked together through an esterbond, an amide bond, and a urea bond;

(2) a poly (ester urethane urea) wherein at least one diol and at leastone amino acid are linked together through an ester bond, a urethanebond, and a urea bond;

(3) a poly (ester amide urethane urea) wherein at least one diol, atleast one diacid, and at least one amino acid are linked togetherthrough an ester bond, an amide bond, a urethane bond, and a urea bond;

(4) a poly (ester amide urethane) wherein at least one diol, at leastone diacid, and at least one amino acid are linked together through anester bond, an amide bond, and a urethane bond;

(5) a poly (ester urea) wherein at least one diol and at least one aminoacid are linked together through an ester bond and a urea bond (in otherwords, at least one diol, a carbonic acid, and at least one amino acidare linked together through an ester bond and a urea bond); and

(6) a poly (ester urethane) wherein at least one diol and at least oneamino acid are linked together through an ester bond and a urethanebond,

further wherein

the at least one diol is a compound of formula HO—R₁—OH, wherein R₁ ischosen from C₁-C₁₂ alkylene optionally interrupted by at least oneoxygen, C₃-C₈ cycloalkylene, C₃-C₁₀ cycloalkylalkylene,

the at least one diacid is a compound of formula HO—(CO)—R₃—(CO)—OH,wherein R₃ is chosen from C₁-C₁₂ alkylene; and

the at least one amino acid is a naturally occurring amino acid or anon-naturally occurring amino acid, such as an L-amino acid or D-aminoacid, further such as L-glycine, L-alanine, L-valine, L-leucine,L-isoleucine, L-proline, L-methionine, L-phenylalanine, or L-tryptophan,or D isomers thereof,

wherein the first polymer and the second polymer are not the same.

In some embodiments of the polymer blend, R₁ for the first polymer ischosen from C₂-C₁₂ alkylene optionally interrupted by at least oneoxygen, C₃-C₈ cycloalkylene, C₃-C₁₀ cycloalkylalkylene,

In some embodiments of the polymer blend, R₁ for the second polymer ischosen from C₂-C₁₂ alkylene optionally interrupted by at least oneoxygen, C₃-C₈ cycloalkylene, C₃-C₁₀ cycloalkylalkylene,

in the second polymer.

In some embodiments of the polymer blend, R₃ for the first polymer ischosen from C₂-C₁₂ alkylene.

In some embodiments of the polymer blend, R₃ for the second polymer ischosen from C₂-C₁₂ alkylene.

In some embodiments of the polymer blend, the second polymer is apoly(ester amide). In some embodiments of the polymer blend, the secondpolymer is a poly(ester amide), wherein the at least one amino acidincludes L-leucine. In some embodiments of the polymer blend, the secondpolymer is a poly(ester amide), wherein the at least one diol includes1,6-hexanediol. In some embodiments of the polymer blend, the secondpolymer is a poly(ester amide), wherein the at least one diacid includessebacic acid.

In some embodiments of the polymer blend, the second polymer is apoly(ester amide), wherein the at least one diol includes1,6-hexanediol, the at least one diacid includes sebacic acid, and theat least one amino acid includes L-leucine.

In some embodiments of the polymer blend, the second polymer is apoly(ester amide), wherein the at least one amino acid is L-leucine. Insome embodiments of the polymer blend, the second polymer is apoly(ester amide), wherein the at least one diol is 1,6-hexanediol. Insome embodiments of the polymer blend, the second polymer is apoly(ester amide), wherein the at least one diacid is sebacic acid.

In some embodiments of the polymer blend, the second polymer is apoly(ester amide), wherein the at least one diol is 1,6-hexanediol, theat least one diacid is sebacic acid, and the at least one amino acid isL-leucine.

In some embodiments of the polymer blend, the first polymer is apoly(ester urea). In some embodiments of the polymer blend, the firstpolymer is a poly(ester urea), wherein the at least one diol includes1,6-hexanediol. In some embodiments of the polymer blend, the firstpolymer is a poly(ester urea), wherein the at least one diacid includescarbonic acid. In some embodiments of the polymer blend, the firstpolymer is a poly(ester urea), wherein the at least one amino acidincludes L-leucine.

In some embodiments of the polymer blend, the first polymer is apoly(ester urea), wherein the at least one amino acid includesL-leucine, the at least one diol includes 1,6-hexanediol, and the atleast one diacid includes carbonic acid.

In some embodiments of the polymer blend, the first polymer is apoly(ester urea), wherein the at least one diol is 1,6-hexanediol. Insome embodiments of the polymer blend, the first polymer is a poly(esterurea), wherein the at least one diacid is carbonic acid. In someembodiments of the polymer blend, the first polymer is a poly(esterurea), wherein the at least one amino acid is L-leucine.

In some embodiments of the polymer blend, the first polymer is apoly(ester urea), wherein the at least one amino acid is L-leucine, theat least one diol is 1,6-hexanediol, and the at least one diacid iscarbonic acid.

In some embodiments of the polymer blend, the first polymer is apoly(ester urea) and the second polymer is a poly(ester amide). In someembodiments of the polymer blend, the first polymer is a poly(esterurea), wherein the at least one diol includes 1,6-hexanediol, the atleast one diacid includes carbonic acid, and the at least one amino acidincludes L-leucine, and the second polymer is a poly(ester amide). Insome embodiments of the polymer blend, the first polymer is a poly(esterurea) and the second polymer is a poly(ester amide), wherein the atleast one diol includes 1,6-hexanediol, the at least one diacid includessebacic acid, and the at least one amino acid includes L-leucine. Insome embodiments of the polymer blend, the first polymer is a poly(esterurea), wherein the at least one diol includes 1,6-hexanediol, the atleast one diacid includes carbonic acid, and the at least one amino acidincludes L-leucine, and the second polymer is a poly(ester amide),wherein the at least one diol includes 1,6-hexanediol, the at least onediacid includes sebacic acid, and the at least one amino acid includesL-leucine.

In some embodiments of the polymer blend, the first polymer is apoly(ester urea) and the second polymer is a poly(ester amide). In someembodiments of the polymer blend, the first polymer is a poly(esterurea), wherein the at least one diol is 1,6-hexanediol, the at least onediacid is carbonic acid, and the at least one amino acid is L-leucine,and the second polymer is a poly(ester amide). In some embodiments ofthe polymer blend, the first polymer is a poly(ester urea) and thesecond polymer is a poly(ester amide), wherein the at least one diol is1,6-hexanediol, the at least one diacid is sebacic acid, and the atleast one amino acid is L-leucine. In some embodiments of the polymerblend, the first polymer is a poly(ester urea), wherein the at least onediol is 1,6-hexanediol, the at least one diacid is carbonic acid, andthe at least one amino acid is L-leucine, and the second polymer is apoly(ester amide), wherein the at least one diol is 1,6-hexanediol, theat least one diacid is sebacic acid, and the at least one amino acid isL-leucine.

In some embodiments of the polymer blend, the first polymer is apoly(ester urea) and the second polymer is a poly(ester amide), whereinthe poly(ester urea) comprises repeating units of:

and the poly(ester amide) comprises repeating units of:

wherein R₁ is chosen from C₁-C₁₂ alkylene optionally interrupted by atleast one oxygen, C₃-C₈ cycloalkylene, C₃-C₁₀ cycloalkylalkylene,

R₃ is C₁-C₁₂ alkylene,

R₂ and R₄ are independently chosen from the side chains of L- andD-amino acids so that the carbon to which R₂ or R₄ is attached has L orD chirality.

In some embodiments of the polymer blend, the ratio of the first polymerto the second polymer ranges from 0.01:0.99 to 0.99:0.01, such as0.05:0.95 to 0.95:0.05, further such as 0.30:0.70 to 0.70:0.30, andfurther such as 0.4:0.6 to 0.6:0.4.

In some embodiments of the polymer blend, the ratio of the first polymerto the second polymer is 0.4:0.6.

In some embodiments of the polymer blend, the ratio of the first polymerto the second polymer is 0.4:0.6, wherein the first polymer is apoly(ester urea) and the second polymer is a poly(ester amide). In someembodiments of the polymer blend, the ratio of the first polymer to thesecond polymer is 0.4:0.6, wherein the first polymer is a poly(esterurea), wherein the at least one diol is 1,6-hexanediol, the at least onediacid is carbonic acid, and the at least one amino acid is L-leucine,and the second polymer is a poly(ester amide), wherein the at least onediol is 1,6-hexanediol, the at least one diacid is sebacic acid, and theat least one amino acid is L-leucine.

Further provided is a process for preparing a diester,

comprising:

heating a mixture comprising

HO—R₁—OH, at least one acid that is not an amino acid, and cyclohexane,wherein

R₁ is chosen from C₁-C₁₂ alkylene optionally interrupted by at least oneoxygen, C₃-C₈ cycloalkylene, C₃-C₁₀ cycloalkylalkylene,

R₂ and R₄ are independently chosen from the side chains of L- andD-amino acids such that the carbon to which R₂ or R₄ is attached has Lor D chirality. For example, R₂ and R₄ are independently chosen from—CH(CH₃)₂, CH₂CH(CH₃)₂, CH(CH₃)CH₂CH₃, and CH₂C₆H₅; R₂ and R₄ can alsobe independently chosen from (CH₂)₃CH₃ and (CH₂)₃SCH₃, with the carbonsto which they are attached having R or S chirality;

the at least one acid that is not an amino acid is chosen from inorganicand organic acids such as sulfonic, sulfuric, and hydrochloric acids,including toluene sulfonic acid (o-toluene, m-toluene, andp-toluenesulfonic acids and methane sulfonic acid.

In some embodiments, R₁ is chosen from C₂-C₁₂ alkylene optionallyinterrupted by at least one oxygen, C₃-C₈ cycloalkylene, C₃-C₁₀cycloalkylalkylene,

In some embodiments, R₂ and R₄ both are the side chain of L-leucine.

In some embodiments, R₁ is —(CH₂)₆—.

In some embodiments, the at least one acid is p-toluenesulfonic acidmonohydrate.

A person of ordinary skill in the art will appreciate that additionalsteps may be required to prepare a diester wherein R₂ or R₄ mayinterfere with diester formation. For example, protection anddeprotection steps known in the art be utilized when R₂ or R₄ is, forexample, the side chain of a poly-functional amino acid, such asL-arginine, L-aspartic acid, L-cysteine, L-glutamate, L-histidine,L-lysine, L-tyrosine, L-serine, and D isomers thereof.

The process provided herein for preparing a diester utilizescyclohexane, a less toxic organic solvent than toluene or benzene,organic solvents used in some previously disclosed processes forproducing monomers and intermediates for the synthesis of amino acidbased polymers. Cyclohexane demonstrates similar azeotrope properties tobenzene under the reaction conditions described herein.

Further provided is a process of preparing the polymers disclosedherein, comprising:

a. mixing a salt of the diester and at least one base in water;

b. mixing at least two bis-electrophiles in an organic solvent;

c. mixing the mixtures from step a and b and stirring vigorously; and

d. obtaining the organic layer including the polymer disclosed herein,

wherein the at least two bis-electrophiles are

a mixture of diacid chloride of formula Cl(CO)—R₃—(CO)Cl andtri-phosgene with a molar ratio of the diacid chloride:triphosgeneranging from 0.95:(0.05/3) to 0.05:(0.95/3) for preparing poly(esteramide urea), or

a mixture of dichloroformate of formula Cl(CO)—O—R₅—O—(CO)Cl andtriphosgene with molar ratio of the dichloroformate:triphosgene rangingfrom 0.95:(0.05/3) to 0.05:(0.95/3) for preparing poly(ester urethaneurea), or

a mixture of diacid chloride of formula Cl(CO)—R₃—(CO)Cl,di-chloroformate of formula Cl(CO)—O—R₅—O—(CO)Cl, and tri-phosgene withmolar ratio of the diacid chloride:dichloroformate:triphosgene rangingfrom 0.90:0.05:(0.05/3) to 0.05:0.05:(0.90/3) for preparing poly(esteramide urethane urea), or

a mixture of diacid chloride of formula Cl(CO)—R₃—(CO)Cl anddi-chloroformate of formula Cl(CO)—O—R₅—O—(CO)Cl with molar ratio of thediacid chloride:dichloroformate ranging from 0.95:0.05 to 0.05:0.95 forpreparing poly(ester amide urethane), wherein R₁ and R₅ areindependently chosen from C₁-C₁₂ alkylene optionally interrupted by atleast one oxygen, C₃-C₈ cycloalkylene, C₃-C₁₀ cycloalkylalkylene,

and

R₃ is C₁-C₁₂ alkylene.

In some embodiments, R₁ and R₅ are independently chosen from C₂-C₁₂alkylene optionally interrupted by at least one oxygen, C₃-C₈cycloalkylene, C₃-C₁₀ cycloalkylalkylene,

In some embodiments, R₃ is C₂-C₁₂ alkylene.

In some embodiments, the organic solvent is chloroform, dichloromethane,or ethyl acetate. In some embodiments, the at least one base is aninorganic base, such as sodium carbonate. In some embodiments, theorganic solvent is amylene-stabilized. For example, in some embodiments,the chloroform, dichloromethane, or ethyl acetate is amylene-stabilized.

In some embodiments, the organic layer obtained in step d is furtherwashed with water one or more times.

In some embodiments, the organic layer obtained after the organic layerobtained in step d is further washed with water one or more times may beused directly for preparing the second composition described belowwithout separation of the polymer.

Alternatively, the poly (ester amide urea) can be prepared by a processcomprising

a. mixing triphosgene, diacid HO(CO)—R₃—(CO)OH, and at least one organicbase in an organic solvent,

b. mixing a salt of the diester and at least one base in water,

c. mixing the mixtures from step a and b and stirring vigorously, and

d. obtaining an organic layer, wherein R₃ is the same as defined above,

wherein the diester has the following formula:

wherein

R₁ is chosen from C₁-C₁₂ alkylene optionally interrupted by at least oneoxygen, C₃-C₈ cycloalkylene, C₃-C₁₀ cycloalkylalkylene,

R₃ is C₁-C₁₂ alkylene; and

R₂ and R₄ are independently chosen from the side chains of L- andD-amino acids such that the carbon to which R₂ or R₄ is attached has Lor D chirality. For example, R₂ and R₄ are independently chosen from—CH(CH₃)₂, CH₂CH(CH₃)₂, CH(CH₃)CH₂CH₃, and CH₂C₆H₅; R₂ and R₄ can alsobe independently chosen from (CH₂)₃CH₃ and (CH₂)₃SCH₃, with the carbonsto which they are attached having R or S chirality.

In some embodiments, R₁ is chosen from C₂-C₁₂ alkylene optionallyinterrupted by at least one oxygen, C₃-C₈ cycloalkylene, C₃-C₁₀cycloalkylalkylene,

In some embodiments, R₃ is C₂-C₁₂ alkylene.

In some embodiments, the salt of the diester is p-toluenesulfonic acidsalt of the diester. In some embodiments, the salt of the diester is ap-toluenesulfonic acid salt of bis-(L-leucine)-1,6-hexylene diester. Insome further embodiments, the p-toluenesulfonic acid salt ofbis-(L-leucine)-1,6-hexylene diester is prepared by direct condensationof L-leucine with 1,6-hexanediol in the presence of p-toluenesulfonicacid monohydrate in refluxed cyclohexane, wherein the ratio of L-leucineto 1,6-hexanediol to p-toluenesulfonic acid monohydrate is 2:1:X,wherein X is greater than 2.

In some embodiments, the at least one base is an inorganic base, such assodium carbonate. In some embodiments, the at least one organic base ispyridine.

In some embodiments, the organic solvent is chloroform, dichloromethane,or ethyl acetate. In some embodiments, the organic solvent isamylene-stabilized. For example, in some embodiments, the chloroform,dichloromethane, or ethyl acetate is amylene-stabilized.

In some embodiments, the poly (ester amide urea) is retained in theorganic layer obtained in step d.

In some embodiments, the organic layer obtained in step d is furtherwashed with water at least one time.

In some embodiments, the organic layer obtained after the organic layerobtained in step d is further washed with water one or more times may beused directly for preparing the second composition described belowwithout separation of the polymer.

In some embodiments, mixing the mixtures from step a and b and stirringvigorously results in interfacial polycondensation. In some embodiments,the by-products of the interfacial polycondensation are highly watersoluble and are retained in water phase. In some embodiments, theby-products of the interfacial polycondensation include sodium chlorideand sodium p-toluenesulfonate.

Alternatively, the poly (ester urethane urea) can be prepared by aprocess comprising

a. mixing triphosgene, diol HO—R₅—OH, and at least one organic base inan organic solvent,

b. mixing a salt of the diester and at least one base in water,

c. mixing the mixtures from step a and b and stirring vigorously, and

d. obtaining an organic layer,

wherein

the diester has the following formula:

wherein

R₁ and R₅ are independently chosen from C₁-C₁₂ alkylene optionallyinterrupted by at least one oxygen, C₃-C₈ cycloalkylene, C₃-C₁₀cycloalkylalkylene,

and

R₂ and R₄ are independently chosen from the side chains of L- andD-amino acids such that the carbon to which R₂ or R₄ is attached has Lor D chirality. For example, R₂ and R₄ are independently chosen from—CH(CH₃)₂, CH₂CH(CH₃)₂, CH(CH₃)CH₂CH₃, and CH₂C₆H₅; R₂ and R₄ can alsobe independently chosen from (CH₂)₃CH₃ and (CH₂)₃SCH₃, with the carbonsto which they are attached having R or S chirality.

In some embodiments, R₁ and R₅ are independently chosen from C₂-C₁₂alkylene optionally interrupted by at least one oxygen, C₃-C₈cycloalkylene, C₃-C₁₀ cycloalkylalkylene,

In some embodiments, the salt of the diester is p-toluenesulfonic acidsalt of the diester. In some embodiments, the salt of the diester is ap-toluenesulfonic acid salt of bis-(L-leucine)-1,6-hexylene diester. Insome further embodiments, the p-toluenesulfonic acid salt ofbis-(L-leucine)-1,6-hexylene diester is prepared by direct condensationof L-leucine with 1,6-hexanediol in the presence of p-toluenesulfonicacid monohydrate in refluxed cyclohexane, wherein the ratio of L-leucineto 1,6-hexanediol to p-toluenesulfonic acid monohydrate is 2:1:X,wherein X is greater than 2.

In some embodiments, the at least one base is an inorganic base, such assodium carbonate. In some embodiments, the at least one organic base ispyridine.

In some embodiments, the organic solvent is chloroform, dichloromethane,or ethyl acetate. In some embodiments, the organic solvent isamylene-stabilized. For example, in some embodiments, the chloroform,dichloromethane, or ethyl acetate is amylene-stabilized.

In some embodiments, the poly (ester urethane urea) is retained in theorganic layer obtained in step d.

In some embodiments, the organic layer obtained after the organic layerobtained in step d is further washed with water one or more times may beused directly for preparing the second composition described belowwithout separation of the polymer.

In some embodiments, the organic layer obtained in step d is furtherwashed with water at least one time.

In some embodiments, mixing the mixtures from step a and b and stirringvigorously results in interfacial polycondensation. In some embodiments,the by-products of the interfacial polycondensation are highly watersoluble and are retained in water phase. In some embodiments, theby-products of the interfacial polycondensation include sodium chlorideand sodium p-toluenesulfonate.

Poly (ester urea) can be prepared by a process comprising

a. mixing a salt of the diester and at least one base in water,

b. mixing triphosgene in an organic solvent,

c. mixing the mixtures from step a and b and stirring vigorously, and

d. obtaining an organic layer including the poly (ester urea),

wherein

the diester has the following formula:

wherein

R₁ is chosen from C₁-C₁₂ alkylene optionally interrupted by at least oneoxygen, C₃-C₈ cycloalkylene, C₃-C₁₀ cycloalkylalkylene,

and

R₂ and R₄ are independently chosen from the side chains of L- andD-amino acids such that the carbon to which R₂ or R₄ is attached has Lor D chirality. For example, R₂ and R₄ are independently chosen from—CH(CH₃)₂, CH₂CH(CH₃)₂, CH(CH₃)CH₂CH₃, and CH₂C₆H₅; R₂ and R₄ can alsobe independently chosen from (CH₂)₃CH₃ and (CH₂)₃SCH₃, with the carbonsto which they are attached having R or S chirality.

In some embodiments, R₁ is chosen from C₂-C₁₂ alkylene optionallyinterrupted by at least one oxygen, C₃-C₈ cycloalkylene, C₃-C₁₀cycloalkylalkylene,

In some embodiments, the salt of the diester is p-toluenesulfonic acidsalt of the diester. In some embodiments, the salt of the diester is ap-toluenesulfonic acid salt of bis-(L-leucine)-1,6-hexylene diester. Insome further embodiments, the p-toluenesulfonic acid salt ofbis-(L-leucine)-1,6-hexylene diester is prepared by direct condensationof L-leucine with 1,6-hexanediol in the presence of p-toluenesulfonicacid monohydrate in refluxed cyclohexane, wherein the ratio of L-leucineto 1,6-hexanediol to p-toluenesulfonic acid monohydrate is 2:1:X,wherein X is greater than 2.

In some embodiments, the organic solvent is chloroform, dichloromethane,or ethyl acetate. In some embodiments, the at least one base is aninorganic base, such as sodium carbonate. In some embodiments, theorganic solvent is amylene-stabilized. For example, in some embodiments,the chloroform, dichloromethane, or ethyl acetate is amylene-stabilized.

In some embodiments, the organic layer obtained in step d is furtherwashed with water.

In some embodiments, the organic layer obtained after the organic layerobtained in step d is further washed with water one or more times may beused directly for preparing the second composition described belowwithout separation of the polymer.

Processes for preparing polymers described herein may be completed morequickly than some previously disclosed processes for preparing aminoacid based polymers. As a non-limiting example, some previouslydisclosed amino acid based polymers required 14 to 16 hours tosynthesize via solution polycondensation, whereas synthesis of a polymeras described herein may be completed in 15 to 20 minutes.

In some embodiments, processes for preparing polymers described hereindo not utilize harmful organic solvents such benzene and toluene, whichare required for the synthesis of some previously disclosedbiodegradable amino acid based polymers.

In some embodiments, processes for preparing polymers described hereindo not utilize organic solvents such as DMF, DMA, and DMSO, which arerequired for the synthesis of some previously disclosed biodegradableamino acid based polymers. DMF, DMA, and DMSO are not compatible withsome bioactive agents, including bacteriophage.

In some embodiments, processes for preparing polymers described hereinutilize cost-effective and readily-purchasable reagents such as sebacoylchloride and triphosgene.

In some embodiments, amino acid based polymers prepared by processesdescribed herein do not need to be separated from the resulting reactionsolution prior to preparing a composition comprising the polymer and atleast one bioactive agent, such as bacteriophage.

In some embodiments, amino acid based polymers prepared by processesdescribed herein do not require purification prior to preparing acomposition comprising the polymer and at least one bioactive agent,such as bacteriophage.

In addition, the polymers described herein may be suitable for preparingbacteriophage-containing compositions for use in wound healing becausepolymers described herein can be solubilized in chloroform, an organicsolvent that does not inactivate bacteriophages when exposure thereto isrelatively short, and the ultimate products of their degradation (carbondioxide, α-amino acids, and neutral fatty diols) can be normal productsof human metabolism. Additionally, the ultimate products of theirdegradation may activate macrophages to produce growth factors thatcould accelerate and improve wound healing. This is in contrast to manywound dressing materials (e.g., poly(lactide/glycolide) copolymers) thatdegrade into acidic products that may be harmful to bacteriophages andmammalian cells. Degradation of the polymers described herein may resultfrom hydrolysis of ester bonds in the polymer backbone.

Polymers provided herein can be formed as non-woven porous materials. Asa non-limiting example, salt leaching may be used to prepare polymericfilms of high porosity. The non-woven porous polymeric materials can beapplied to wounds in place of a gauze. In some embodiments, a non-wovenporous polymeric material of the disclosure is soaked in liquidbacteriophage and used as a wound dressing.

In some embodiments, the non-woven porous polymeric material mayexpedite wound healing.

In some embodiments, the non-woven porous polymeric material adheres tothe wound site. In some embodiments, adherence of the non-woven porouspolymeric material to the wound site results in at least partialsuppression of inflammation.

Non-woven porous or non-porous polymeric materials provided herein (asdescribed above or below) do not require removal from the wound sitebecause the materials are biodegradable. In some embodiments, thenon-woven porous or non-porous polymeric material may be at leastpartially degraded during the wound healing process. For example, thenon-woven porous polymeric material may be 0.1%, 0.5%, 1%, 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 99%, 99.5%, 99.9% or 100% degraded during the wound healingprocess.

In some embodiments, the non-woven porous polymeric material may becompletely degraded during the wound healing process.

Also provided herein is a process for preparing a non-woven porouspolymeric material comprising the steps of:

a. mixing a polymer of the disclosure in a mixture comprising at leastone salt and an organic solvent;

b. casting the resulting mixture from step a onto a hydrophobic surface;

c. evaporating the organic solvent to obtain a film; and

d. leaching the at least one salt from the film.

In some embodiments, the organic solvent is chloroform, dichloromethane,or ethyl acetate.

In some embodiments, the salt is sodium chloride.

In some embodiments, the mixture is cast between two glass plates anddried for approximately 24 hours.

In some embodiments, drying occurs due to solvent evaporation.

In some embodiments, salt leaching occurs as a result of immersing thefilm in water for an effective period of time (e.g., 1, 12, 24, or 36hours).

Compositions

Bacteriophages are polyelectrolytes that have an internal charge dipole.

Provided herein is a first composition in a powdery form comprising atleast one bacteriophage.

Also provided herein is a first composition comprising at least onebacteriophage and at least one inorganic salt. In some embodiments, thecomposition is in the form of dry powder.

Inorganic salts are generally considered to be non-immunogenic. As aresult, compositions comprising at least one bacteriophage and at leastone inorganic salt may be less likely to provoke an immune response in apatient than previously published phage delivery technologies, whichgenerally utilized phage stabilizing additives that may be immunogenic.As a non-limiting example, gelatin may cause an immune response in somepatients.

In some embodiments, the at least one inorganic salt is selected frominorganic salts having poor water solubility, such as calcium salts,magnesium salts, strontium salts, and barium salts, and particularlysuch as calcium salts and magnesium salts.

In some embodiments, the at least one inorganic salt is selected fromcalcium carbonate, calcium phosphate, magnesium carbonate, and magnesiumphosphate.

Compositions comprising at least one bacteriophage and at least oneinorganic salt may protect bacteriophage, which are known to bepH-sensitive, when the compositions are used to treat wounds. As anon-limiting example, a composition comprising at least onebacteriophage and at least one poorly soluble carbonate salt may protectbacteriophage in wound environments that are more acidic than thesurrounding tissue. As a result, compositions comprising at least onebacteriophage and at least one inorganic salt may be moretherapeutically effective than compositions comprising at least onebacteriophage and no inorganic salts to buffer the wound environment.

In some embodiments, the at least one inorganic salt is a mixture ofMgCO₃ and CaCO₃. In some embodiments, the weight ratio of MgCO₃ to CaCO₃ranges from 5:95 to 95:5, such as the ratio is 5:95. The at least oneinorganic salt such as calcium and magnesium salts may, in someembodiments, positively influence wound healing by stabilizing andactivating the bacteriophage.

In some embodiments, the first composition further comprises at leastone other bioactive agent. In some embodiments, the at least one otherbioactive agent is selected from: antiseptics, anti-infectives,antibiotics, pain relievers, antibacterials, antiprotozoal agents, andantiviral agents, analgesics, anti-inflammatory agents includingsteroids and non-steroidal anti-inflammatory agents including COX-2inhibitors and anti-neoplastic agents, contraceptives, CNS active drugs,hormones, enzymes, hemostatics, proteases, collagenases, and vaccines.Examples of those bioactive agents can be found in other parts of thisdisclosure. In some embodiments, the first composition is in the form ofa spray or patch or film. In other embodiments, the first composition isin the form of a gel or an ointment.

The first composition in dry powder form comprising at least onebacteriophage and at least one inorganic salt can be used to treatinfected wounds and cavities. These preparations could also be used totreat osteomyelitis and to fill/reconstruct bone tissues, and could beused in a variety of dental products.

The first composition can be also used in food processing to providefood safety. For example, CaCO₃ and Ca₃(PO₄)₂ with adsorbed phages(against Salmonella, Escherichia coli, etc.) can be very useful in foodprocessing since the salts themselves are widely used as food additives.

The first composition can be also used in livestock against pathogenicbacteria, e.g. pathogenic E. coli, Salmonella, etc. These preparationscan be added to feed, and carbonate salts can protect the phages frominactivation by the action of acidic media of gastric juice. The firstcomposition can further be used in agriculture to treat plants.

Further provided is a process for preparing the first composition,comprising:

mixing and holding (incubating) the at least one inorganic salt and theat least one bacteriophage;

filtrating the suspension obtained to produce the at least onebacteriophage adsorbed (immobilized) wet solid product;

washing the obtained wet solid product with saline solution optionally;and

drying the obtained wet solid product through vacuum drying, freezedrying, lyophilization, or spray-drying to obtain the first composition.

In some embodiments, the at least one salt is selected from inorganicsalts as disclosed herein. In some embodiments, the at least one saltand at least one bacteriophage in the form of liquid is mixed in anappropriate w/v (g/mL) ratio such as a ratio of 1:10. In someembodiments, the process for preparing the first composition is carriedout at room temperature and under sterile conditions.

Also provided is a first composition comprising at least onebacteriophage and components from buffer. In some embodiments, the firstcomposition is in a powdery form. In some embodiments, the buffercomprises at least one inorganic salt. In some embodiments, the bufferis TMN (Tris-MgCl₂—NaCl) buffer. In some embodiments, the buffer isDulbecco's Phosphate Buffered Saline with MgCl₂ and CaCl₂. Also providedis a process for preparing the first composition comprising 1) mixing atleast one bacteriophage and at least one buffer, and 2) drying themixture through vacuum drying, freeze drying, lyophilization, orspray-drying. In some embodiments, the mixture is dried through freezedrying.

Further provided is a second composition comprising at least one polymerdescribed herein, at least one bioactive agent, and, in someembodiments, at least one filler. For example, in some embodiments ofthe second composition, the at least one polymer is an amino acid basedpolymer as described herein. In some embodiments of the secondcomposition, the at least one polymer is a polymer blend as describedherein. The second composition may be applied to an internal or externalsurface of the body to deliver an effective amount of the at least onebioactive agent.

In some embodiments of the second composition, the bioactive agent isselected from bacteriophage and phage product. Non-limiting examples ofphage product include endolysin, phage proteins, and phage enzymes.

In some embodiments of the second composition, the bioactive agentcomprises one or more of an antiseptic, an anti-infective (e.g., abacteriophage), a bacteriophage-derived product (e.g., endolysin, phageprotein, or phage enzyme), an antibiotic, an antibacterial, anantiprotozoal agent, an antiviral, an analgesic, an anti-inflammatoryagent (e.g., steroids or non-steroidal anti-inflammatory agents such asCOX-2 inhibitors), an anti-neoplastic agent, a contraceptive, a centralnervous system (CNS) active drug, an hormone, an enzyme, or a vaccine.

In some embodiments of the second composition, the bioactive agentcomprises one or more of a phage stabilizing additive, a fibrinolyticenzyme, a metabolic process stimulating agent, a vasodilator, a painkiller, mono- and disaccharides, polysaccharides andmucopolysaccharides, an anti-protozoa drug, an anti-fungal drug, ahemostatic, a vitamin, an anti-inflammatory steroid, or ananti-inflammatory non-steroid drug.

Non-limiting examples of the enzymes include those that can catalyze thehydrolysis (erosion) of the polymer disclosed herein. The hydrolysis(erosion) of the polymers disclosed herein can be important for therelease of the at least one bioactive agent into the surroundingtissues. At least one enzyme may also be used, as a non-limitingexample, to treat wounds and abrasions by removing the dead or infectedskin from the site of injury. Non-limiting examples of the at least oneenzyme include papain, collagenase, elastase, fibrinolysin,hyaluronidase, trypsin, α-chymotrypsin and lipase. In some embodimentsof the second composition, the at least one enzyme is selected fromtrypsin, α-chymotrypsin and lipase.

Non-limiting examples of antibiotics include fluoroquinolones (e.g.,tetracycline, ciprofloxacin, and levofloxacin), monoxycarbolic acidantibiotics (e.g., mupirocin), aminoglycosides (e.g., neomycin),macrolide antibiotics (e.g., erythromycin), bacitracin, Polymyxin, andmixtures thereof. Additional non-limiting examples of antibioticsinclude silver salts (e.g., silver sulfadiazine and silver nitrate),chlorohexidine, and mafenide acetate.

A non-limiting example of a phage stabilizing additive is calciumgluconate.

Non-limiting examples of fibrinolytic enzymes include hyaluronidase andfibrinolysin.

A non-limiting example of a metabolic process simulating agent ismethyluracyl.

Non-limiting examples of vasodilators include sodium hydrocarbonate andL-arginine.

Exemplary pain relievers include, but are not limited to, benzocaine,lidocaine, tetracaine, pramocaine, dibucaine, and mixtures thereof.

A non-limiting example of an anti-protozoa drug is metronidazole.

A non-limiting example of an anti-fungal drug is clotrimazolum.

A non-limiting example of a hemostatic is thrombin.

A non-limiting example of an anti-inflammatory steroid is prednisolone.

A non-limiting example of an anti-inflammatory non-steroid drug issodium diclofenac (Voltaren).

In some embodiments of the second composition, the second compositioncomprises at least one antibiotic selected from silver salts (e.g.,silver sulfadiazine and silver nitrate), chlorohexidine, and mafenideacetate.

In some embodiments of the second composition, the second compositioncomprises calcium gluconate as a phage stabilizing additive.

In some embodiments of the second composition, the second compositioncomprises at least one fibrinolytic enzyme selected from hyaluronidaseand fibrinolysin. In some embodiments, the composition comprisesmethyluracyl as a metabolic process simulating agent.

In some embodiments of the second composition, the second compositioncomprises sodium hydrocarbonate or L-arginine as a vasodilator.

In some embodiments of the second composition, the second compositioncomprises benzocaine.

In some embodiments of the second composition, the second compositioncomprises metronidazole.

In some embodiments of the second composition, the second compositioncomprises clotrimazolum.

In some embodiments of the second composition, the second compositioncomprises thrombin.

In some embodiments of the second composition, the second compositioncomprises prednisolone.

In some embodiments of the second composition, the second compositioncomprises sodium diclofenac.

In some embodiments of the second composition, the at least one filleris selected from inorganic salts, sucrose, and gelatin. In someembodiments of the second composition, the inorganic salts includecalcium salts, magnesium salts, strontium salts, and barium salts. Insome embodiments of the second composition, the at least one filler isselected from calcium salts and magnesium salts. In some embodiments ofthe second composition, the at least one filler is selected from calciumcarbonate, calcium phosphate, magnesium carbonate, and magnesiumphosphate. In some embodiments of the second composition, the at leastone filler is selected from calcium carbonate and magnesium carbonate.In some embodiments, the at least one filler is a mixture of calciumcarbonate and magnesium carbonate.

In some embodiments of the second composition, the at least one polymeris selected from poly (ester amide urea), poly (ester urethane urea),poly (ester amide urethane urea), and poly (ester amide urethane).

In some embodiments, the second composition comprises at least onepolymer selected from poly (ester amide urea), poly (ester urethaneurea), poly (ester amide urethane urea), and poly (ester amideurethane), at least one bacteriophage or phage product, calciumcarbonate, magnesium carbonate, benzocaine, ciprofloxacin, and an enzymesuch as chymotrypsin. In some embodiments, the second compositioncomprises a poly (ester amide urea), at least one bacteriophage or phageproduct, calcium carbonate, magnesium carbonate, benzocaine,ciprofloxacin, and an enzyme such as chymotrypsin.

In some embodiments of the second composition, the at least one polymeris a polymer blend as described herein. In some embodiments of thesecond composition, the at least one polymer is a polymer blend whereinthe first polymer is a poly(ester urea) and the second polymer is apoly(ester amide). In some embodiments of the second composition, thefirst polymer is a poly(ester urea), wherein the at least one diol is1,6-hexanediol, the at least one diacid is carbonic acid, and the atleast one amino acid is L-leucine, and the second polymer is apoly(ester amide), wherein the at least one diol is 1,6-hexanediol, theat least one diacid is sebacic acid, and the at least one amino acid isL-leucine.

In some embodiments of the second composition, the second compositioncomprises a poly(ester urea), a poly(ester amide), at least onebacteriophage or phage product, calcium carbonate, magnesium carbonate,benzocaine, ciprofloxacin, and an enzyme such as chymotrypsin.

In some embodiments of the second composition, the at least one polymeris grindable. In some embodiments, the second composition comprising agrindable polymer is in the form of fine powder suitable for applicationin a spray wound dressing.

The second composition described herein may also possess desirablemechanical properties for wound dressings, including tissue-likeelasticity. In some embodiments, the second composition described hereinmay possess sufficient plasticity to form a film, which can be manuallydeformed to fit tightly to an irregular biological surface (e.g., aconcave wound surface).

In some embodiments of the second composition, the second composition isin the form of a perforated film, a patch, a film or a spray. In otherembodiments of the second composition, the second composition is in theform of an unperforated film, a gel, a hydrogel, or an ointment. In someembodiments of the second composition, the film form of the secondcomposition may be a single layer or multiple layers. The person skilledin this art will appreciate that patches and perforated films can be ofany practical dimension. Additionally, the patches and perforated filmsdisclosed herein can be designed in virtually any size or shape, as maybe useful for one or more specific applications. Additionally, filmsmade from the second composition described herein may be readilyseparable by gentle manual force, desirably leaving each sheet of filmintact upon separation.

In some embodiments of the second composition, the second composition isin the form of a non-woven porous material. As a non-limiting example,salt leaching may be used to prepare non-woven porous materials.

In some embodiments of the second composition, the second composition inthe form of a non-woven porous material adheres to the wound site. Insome embodiments, adherence of the non-woven porous material to thewound site results in at least partial suppression of inflammation.

Also provided herein is a process for preparing a non-woven porouspolymeric material comprising the steps of:

a. mixing a polymer of the disclosure in a mixture comprising at leastone salt and an organic solvent;

b. casting the resulting mixture from step a onto a hydrophobic surface;

c. evaporating the organic solvent to obtain a film; and

d. leaching the at least one salt from the film.

In some embodiments of the second composition, the second compositionmay be used to provide a coating on a support material, which may or maynot be biodegradable, such as a fibrous or non-fibrous three-dimensionalconstruct or a woven support. Constructs prepared with the secondcomposition may be part of devices including a support material to beused as, for example, bandages for wounds or burn dressings.

In some embodiments, constructs comprising the second composition may besurgically implanted. Constructs according to the present disclosure mayalso be formed into devices for wound packing, such as gel foams, or maybe used as components in surgical appliances, such as Penrose drains,indwelling catheters, catheters for peritoneal dialysis, and any otherappliances that are in contact with body cavities, the bloodcirculation, or the lymphatic circulation and are used to treat bothinfection and potential infections.

Additional non-limiting embodiments include constructs for oral hygienesuch as gum implants (e.g., for periodontal disease or dental caries).Such constructs may contain at least one or more bioactive agentreleased in a controlled manner upon erosion of the construct. Suitableselections of particular bioactive agents and effective amounts thereofwill be readily apparent to the person skilled in the art in view of theintended site of implantation.

Further provided is a method of treating a patient having an ulcerativewound comprising inserting into the wound or covering the wound with thesecond composition as disclosed herein.

In some embodiments, the wound is open or infected.

In some embodiments, the at least one bacteriophage in the secondcomposition is specific for bacteria found in the wound.

Further provided is a process for preparing the second composition,comprising:

a. mixing the first composition described herein with a mixturecomprising an organic solvent and at least one polymer disclosed herein;optionally adding at least one another bioactive agent;

b. casting the resulting mixture from step a onto a hydrophobic surface;and

c. removing the organic solvent to obtain a film containing the secondcomposition.

In some embodiments, the organic solvent is dichloromethane.

In some embodiments, the mixture comprises 13% w/v polymer.

In some embodiments, mixing in step a is performed slowly at a speed of100 rpm or lower.

In some embodiments, mixing in step a is accomplished using a frictiontype homogenizer with pestle at a speed of 100 rpm or lower. In someembodiments, mixing in step a occurs for 10 minutes.

In some embodiments, the hydrophobic surface is a Teflon petri dish.

In some embodiments, removal of the organic solvent occurs viaevaporation. In some embodiments, removal of the organic solvent occursvia evaporation over the course of four days.

In some further embodiments, a vacuum-drier is used to remove excesssolvent. In some embodiments, the vacuum-drier operates at 37° C.

Alternatively, provided is a process for preparing the secondcomposition, comprising

a. mixing a liquid comprising at least one bacteriophage with a mixturecomprising an organic solvent and at least one polymer disclosed herein;optionally adding at least one filler selected from the inorganic saltsdisclosed above and at least one another bioactive agent;

b. casting the resulting mixture from step a onto a hydrophobic surface;and

c. removing the organic solvent to obtain a film containing the secondcomposition.

In some embodiments, the organic solvent is chloroform. In otherembodiments, the organic solvent is dichloromethane or ethyl acetate. Insome embodiments, the mixture comprising an organic solvent and at leastone polymer further comprises additional bioactive agent chosen fromantiseptics, anti-infectives, such as bacteriophages, antibiotics,antibacterials, antiprotozoal agents, and antiviral agents, analgesics,anti-inflammatory agents including steroids and non-steroidalanti-inflammatory agents including COX-2 inhibitors, anti-neoplasticagents, contraceptives, CNS active drugs, hormones, hemostatics,enzymes, proteases, and vaccines.

Alternatively, provided herein in a process for preparing the secondcomposition comprising

a. mixing the at least one bioactive agent with a mixture comprising anorganic solvent and at least one polymer described herein;

b. casting the resulting mixture from step a onto a hydrophobic surface;and

c. removing the organic solvent to obtain a film.

In some embodiments, the second composition comprises poly (ester amideurea), at least one or more bacteriophage, calcium carbonate, magnesiumcarbonate, benzocaine, ciprofloxacin, and chymotrypsin.

Also provided herein is a wound dressing comprising the firstcomposition or the second composition disclosed herein.

Also provided herein is an implantable surgical device comprising thefirst composition or the second composition disclosed herein.

Also provided herein is a food or animal feed additive comprising thefirst composition or the second composition disclosed herein.

Also provided herein is a method of treating agricultural cropscomprising administering the first composition or the second compositiondisclosed herein. In some embodiments, the first composition or thesecond composition is administered by spraying the composition on theagricultural crops.

EXAMPLES

The present application refers to a number of documents, the contents ofwhich are hereby incorporated by reference to the extent they disclosesuitable, conventional methods known to those skilled in the field.Also, the present application claims priority from U.S. patentapplication Ser. No. 15/188,783 filed Jun. 21, 2016, which is alsohereby incorporated by reference in its entirety.

The following examples are intended for illustration purposes only andshould not be construed as limiting the scope of the disclosure or theclaims appended hereto in any way.

All exemplary preparations should be performed under sterile conditionswith sterile glass-ware and storage vessels. In addition, a chemicalhood with good ventilation or a biological hood should be used whenperforming exemplary preparations described below.

Example 1: Preparation of Bis-(Alpha-Amino Acid)-Alkylene Diester:General Procedure

A mixture of

(R2 and R4 are defined as before, which can be the same or different,both amino acids together are 2 moles), HO—R1—OH (R1 is defined asbefore, 1 mole), and p-TsOH monohydrate (2 moles) is refluxed incyclohexane.

Examples for

and include L-amino acids, such as L-leucine.

Examples for HO—R₁—OH can be any aliphatic diol, including α,ω-alkylenediols like HO—(CH₂)_(k)—OH (i.e. non-branched), branched diols (e.g.,1,2-propylene glycol), cyclic diols (e.g. dianhydrohexitols andcyclohexanediol), or oligomeric diols based on ethylene glycol such asdiethylene glycol, triethylene glycol, tetraethylene glycol, orpoly(ethylene glycol)s). A further example of HO—R₁—OH can be1,6-hexanediol.

One exemplary diester is bis(L-leucine)-1,6-hexylene diester with thefollowing structure:

Di-p-toluenesulfonic acid salt of bis-(L-leucine)-1,6-hexylene diestermay be prepared by refluxing a mixture of L-leucine (2 moles),1,6-hexanediol (1 mole), and p-TsOH monohydrate (2 or more moles) incyclohexane.

Example 2: Preparation of Bis-Electrophilic Monomers

A. Diacid Chlorides

Diacid chloride of formula Cl(CO)—R₃—(CO)Cl can be prepared as follows:gently heating (such as heating at 40-50° C.) free di-acids(OH(CO)—R₃—(CO)OH) with excess of chlorinating agent that is thionylchloride, or 2 moles of chlorinating agent what is phosphoruspentachloride PCl₅ (2 moles per 1 mole of di-acid), without using anycatalyst and organic solvent; additional heating at 40-50° C. for 12 hafter complete dissolution of solid diacid; removing the excess thionylchloride or phosphorus oxychloride (POCl₃) at 40-50° C. under reducedpressure. R3 is as defined before.

This procedure can result in pure, polycondensation grade di-acidchlorides which are used in the preparation of the polymers disclosedherein directly, without additional purification.

B. Preparation of Dichloroformate of Diols

Di-chloroformate Cl(CO)—O—R₅—O—(CO)Cl can be prepared as follows: gentlyheating (40-50° C.) the HO—R₅—OH with excess phosgene (6-8 moles per 1mole of diol) in tetrahydrofuran (THF) solution until completedissolution of solid diol; additional heating at 40-50° C. for 12 h;removing THF and excess phosgene at 40-50° C. under reduced pressure.This procedure results in pure, polycondensation grade di-chloroformateswhich are used in the preparation of the polymers disclosed hereindirectly, without additional purification. R₅ is as defined before.

Example 3: Preparation of the Polymers

General Procedures for the Synthesis of the Polymers Via InterfacialPolycondensation

Poly(ester urea), poly(ester urethane), poly(ester amide urea),poly(ester urethane urea), poly(ester amide urethane), and poly(esteramide urethane urea) are prepared using the following protocol.

2.0 mol of di-p-toluenesulfonic acid salt of bis(L-amino acid)-alkylenediester is added to a reactor suitable for interfacial polycondensation.15.0 L of water is then added to the reactor with stirring. To theobtained suspension of di-p-toluenesulfonic acid salt of bis(L-aminoacid)-alkylene diester in water, 6.0 mol of anhydrous sodium carbonateis added with stirring at room temperature for approximately 30-40 min.Hereafter, the aqueous solution of di-p-toluenesulfonic acid salt ofbis(L-amino acid)-alkylene diester and anhydrous sodium carbonate isreferred to as the first solution.

In a separate reaction vessel, 2.0 mol of bis-electrophilic monomer or amixture of at least two bis-electrophilic monomers, totaling 2.0 mol ofbis-electrophilic monomer, is dissolved in 6.5 L of amylene-stabilizedchloroform. Hereafter, the solution of bis-electrophilic monomer inamylene-stabilized chloroform is referred to as the second solution.Other solvents may be used in place of amylene-stabilized chloroform,including but not limited to, amylene-stabilized methylene chloride,dichloromethane, and ethyl acetate, among others.

The second solution is quickly added to the first solution at 15-20° C.to produce a water/organic mixture. The water/organic mixture isvigorously stirred for approximately 15-20 minutes. After the cessationof stirring, the mixture is allowed to separate, resulting in atwo-layer system. The lower layer, comprising polymer in chloroform, isseparated and washed 3 times (3×6 L) with distilled water to remove thesalts (sodium chloride, sodium carbonate/bicarbonate, and sodiump-toluene sulfonate). The chloroform layer is separated after each washstep. After washing, the chloroform layer is dried over anhydrous Na₂SO₄(0.8-1.0 kg) and filtered off. The obtained chloroform layer is storedfor the subsequent applications, including, but not limited to, thepreparation of bacteriophage containing bio-composites.

When preparing poly(ester amide urea), the second solution contains twobis-electrophilic monomers, i.e., a diacid chloride and tri-phosgene, ata molar ratio ranging from 0.95:(0.05/3) to 0.05:(0.95/3).

When preparing poly(ester urethane urea), the second solution contains amixture of two bis-electrophilic monomers, i.e., a dichloroformate of adiol and tri-phosgene, at a molar ratio ranging from 0.95:(0.05/3) to0.05:(0.95/3).

When preparing poly(ester amide urethane urea), the second solutioncontains a mixture of three bis-electrophilic monomers, a diacidchloride, a dichloroformate of a diol, and tri-phosgene, at a molarratio ranging from 0.9:0.05:(0.05/3) to 0.05:0.05:(0.9/3).

When preparing poly(ester amide urethane), the second solution containsa mixture of two bis-electrophilic monomers, i.e., a diacid chloride anda dichloroformate of a diol, at a molar ratio ranging from 0.99:0.01 to0.01:0.99, such as 0.05:0.95 to 0.95:0.05, and further such as 0.10:0.90to 0.90:0.10.

In alternative procedures, the polymer is isolated after thepolycondensation reaction by placing the obtained chloroform layer in aglass vessel and removing the solvent (chloroform) by distillation underatmospheric pressure. As will be recognized by those skilled in the art,“atmospheric pressure” may be relative depending on geographic location,and the exact transition point may vary slightly depending on ambientconditions, for example, geographic location or temperature.

Subsequently, 6.0 Ls of hot water (70-80° C.) are added to the formedmass to obtain a polymer solution. The resulting rubbery polymer isremoved from the glass vessel and placed between plates, which issqueezed to remove residual water. The plates are placed in an ovenequipped with a fan and dried at 100° C. For further drying, the polymeris moved to a vacuum-drier and dried at 100° C. The polymer is weighedat regular intervals and dried until the polymer weight did not changeover the course of multiple weighing times.

A. Poly(Ester Amide Urea)

Di-p-toluenesulfonic acid salt of bis(L-leucine)-1,6-hexylene diester(1.378 g, 2.0 mol) was added to a reactor suitable for interfacialpolycondensation. Water (15.0 L) was added to the reactor with stirring.To the obtained suspension, anhydrous sodium carbonate (0.636 kg, 6.0mol) was added and stirred at room temperature for approximately 30-40min (the first solution). In a separate vessel, tri-phosgene (0.1682 kg,1.7/3 mol) and sebacoyl chloride (i.e., ClCO—(CH₂)₈—COCl, 0.0717 kg, 0.3mol) were dissolved in 6.5 L of amylene-stabilized chloroform (thesecond solution). The second solution was rapidly added to the firstsolution at room temperature to produce a water/organic mixture.

The water/organic mixture was stirred vigorously for about 15-20 min.Following cessation of stirring, the mixture was allowed to separatecompletely, resulting in a two-layer system. The lower layer containingpoly(ester urea amide) in chloroform was separated and washed 3 times(3×6 L) with distilled water to remove the salts (sodium chloride,sodium carbonate/bicarbonate, and sodium p-toluene-sulfonate). Thechloroform layer was separated after each wash step.

After washing, the chloroform layer was separated again, dried overanhydrous Na₂SO₄ (0.8-1.0 kg), and filtered off. The solution was thendiluted up to the desired concentration, 0.6 kg of the polymer in 8.0 Lof chloroform. The filtered solution contained ca. 0.78 kg of thepolymer in ca. 6.5 L of chloroform. To obtain the desirableconcentration (0.6 kg in 8.0 L), 2.75 L of amylene-stabilized chloroformwas added to 5.25 L of the polymer solution. The resulting solutioncontains 0.6 kg of the polymer in 8.0 L chloroform and is ready forpreparing the composition.

In one instance, the polymer was isolated right after thepolycondensation reaction according to the following procedure: theobtained chloroform layer after filtering off Na₂SO₄ was placed in glassvessel and the solvent (chloroform) was removed by distillation underatmospheric pressure (ca. 5.5-6.0 LL chloroform is collected).Afterwards, 6.0 Ls of hot water (ca. 70-80° C.) were added to the formedmass. The resulting rubbery polymer was removed from the glass vesseland placed onto the Teflon® plates, squeezed to remove and pour out theresidual water, the plates was placed in an oven equipped with a fan anddried at 100° C. For a final drying the polymer was moved to avacuum-drier and dried at 100° C. up to constant weight. Yield: 0.743 kg(95%). Mw=45-55 kDa, Polydispersity (Mw/Mn)=1.6-1.8 (gel phasechromatography; a solution of LiBr (0.1 M) in N,N-dimethylformamide wasused as an eluent at a flow rate 1.0 mL/min).

B. Poly(Ester Urea Urethane)

In a similar example, poly(ester urea urethane) can be prepared byinterfacial polycondensation of di-p-toluenesulfonic acid salt ofbis-(L-leucine)-1,6-hexylene diester (1.0 mole) with a mixture ofdi-chloroformate of 1,6-hexanediol/triphosgene at a molar ratio0.15:(0.85/3).

C. One-Pot Synthesis of Poly(Ester Urea Amide)s—General Procedure

2.0 mole of di-p-toluenesulfonic acid salt of bis(L-amino acid)-alkylenediester is placed in a reactor for interfacial polycondensation. 15.0 Lof water is added on stirring. To the obtained suspension 0.636 kg (6.0mol) of anhydrous sodium carbonate is added and stirred at roomtemperature for about 30-40 min (The 1st solution).

In a separate vessel, x mole of dicarboxylic acid (of general formulaHOCO—(CH2)y-COOH) and 2 x mole of dry pyridine in 6.5 L ofamylene-stabilized chloroform (2+5x)/3 mole [2x mole for in situsynthesis of a mole of di-acid chloride which forms (ester amide)blocks+(2−x)/3 mole which forms (ester urea) blocks] of triphosgene isadded and stirred for an appropriate time (The 2nd solution).

To the 1st solution is quickly added the 2nd solution at 15-20° C. andthe water/organic mixture is vigorously stirred for about 15-20 min. Thestirrer is stopped and the mixture is allowed to separate, resulting intwo layer system. The lower layer, containing the polymer in chloroform,is separated, washes 3-times (3×6 L) with distilled water to remove thesalts—sodium chloride, sodium carbonate/bicarbonate, and sodiump-toluene sulfonate; the chloroform layer is separated after eachportion of washing with water. After washing, the chloroform layer isseparated again, dried over anhydrous Na₂SO₄ (0.8-1.0 kg) and filteredoff. The obtained chloroform solution is stored for the subsequent use.

D. One-Pot Synthesis of Poly(Ester Urethane Urea)—General Procedure

2.0 mole of di-p-toluenesulfonic acid salt of bis(L-amino acid)-alkylenediester is placed in a reactor for interfacial polycondensation. 15.0 Lof water is added on stirring. To the obtained suspension is addedanhydrous sodium carbonate (0.636 kg, 6.0 mol) of and stirred at roomtemperature for about 30-40 min (The 1^(st) solution).

In a separate vessel, x mole of alkylenediol (of general formulaHO—(CH₂)_(x)—OH) and 2x mole of dry pyridine in 6.5 L ofamylene-stabilized chloroform (2+x)/3 mole [2x/3 mole for in situsynthesis of x mole bis-chloroformate which forms (ester urethane)blocks+(2−x)/3 mole which forms (ester urea) blocks] of triphosgene wasadded and stirred for an appropriate time (The 2^(nd) solution).

To the 1^(st) solution is added the 2^(nd) solution i quickly at 15-20°C. and the water/organic mixture is vigorously stirred for about 15-20min. The stirrer is stopped and the mixture is allowed to separate,resulting in two layer system. The lower layer, containing the polymerin chloroform, is separated, is washed 3-times (3×6 L) with distilledwater to remove the salts—sodium chloride, sodium carbonate/bicarbonate,and sodium p-toluene sulfonate; the chloroform layer is separated aftereach portion of washing with water. After washing, the chloroform layeris separated again, is dried over anhydrous Na₂SO₄ (0.8-1.0 kg) andfiltered off. The obtained chloroform solution is stored for thesubsequent use.

E. Grindable Poly (Ester Urea)

2.0 mole of di-p-toluenesulfonic acid salt ofbis(L-leucine)-1,6-hexylene diester was placed in a reactor forinterfacial polycondensation. Water (15.0 L) was added to the reactor onstirring. To the obtained suspension was added 0.636 kg (6.0 mol) ofanhydrous sodium carbonate. The resulting mixture was stirred at roomtemperature for about 30-40 min (The 1st solution).

1.9 mole triphosgene (5 mol % deficient as compared withbis(L-leucine)-1,6-hexylene diester above) was dissolved in 6.5 L ofamylene-stabilized chloroform in a separate vessel (The 2nd solution).

To the 1st solution was the 2nd solution quickly at 15-20° C. and thewater/organic mixture was vigorously stirred for about 15-20 min. Thestirrer was stopped and the mixture was allowed to separate, resultingin two layer system. The lower layer, containing poly (ester urea) inchloroform, was separated, washed 3-times (3×6 L) with distilled waterto remove the salts—sodium chloride, sodium carbonate/bicarbonate, andsodium p-toluene sulfonate.

The chloroform layer was separated after each portion of washing withwater. After washing, chloroform layer was separated again, dried overanhydrous Na₂SO₄ (0.8-1.0 kg) and filtered off. The obtained chloroformsolution was stored for the subsequent use. The obtained poly(esterurea) labeled as LMW-1L6 was of low-molecular-weight (M_(w)=5-6 KDa) andwas grindable in solid state.

Example 4: Preparation of Powdery Bacteriophages (the First Composition)

Powdery salts with immobilized bacteriophages were prepared by theincubation of at least one salt in a liquid preparation of at least onebacteriophage and subsequent drying using vacuum-drying, freeze-drying,or spray-drying methods.

The at least one salt (i.e., absorbent) was selected from, for example,MgCO₃, CaCO₃, and Ca₃(PO₄)₂. The average particle size of MgCO₃, CaCO₃,and Ca₃(PO₄)₂ is 200, 50, and 5 μm, respectively.

The at least one bacteriophage was selected from, for example,Staphylococcus aureus, Pyophage (a cocktail of five phages: E. coli,Proteus, Staphylococcus aureus, Streptococcus, and Pseudomonasaeruginosa) and Intestiphage (a cocktail of seven phages: E. coli,Proteus, Staphylococcus aureus, Streptococcus, Pseudomonas aeruginosa,Shigella, and Salmonella). All were prepared by Biochimpharm, LLC,Tbilisi, Ga.

Step 1. Incubation of the Solid Adsorbents and Phages

To 0.3 kg of powdery adsorbent (MgCO₃, CaCO₃, Ca₃(PO₄)₂) was added 3.0 L(w/v ration 1:10) of liquid bacteriophage (Staphylococcus, Pyophage, orIntestiphage) preparation at room temperature. The mixture wasthoroughly homogenized by stirring for 20-30 min. The stirring wasstopped and the mixture was kept at room temperature for an additional30 min without agitation.

Step 2. Filtration of the Obtained Suspension, Obtaining (Wet)“Phage-Adsorbed” Solid Product

The suspension obtained in Step 1 was filtered off under sterileconditions. The filtrates (F) are referred below as Salt/Bacteriophage,F₀:

MgCO₃/Staphylophage, F₀

CaCO₃/Staphylophage, F₀

Ca₃(PO₄)₂/Staphylophage, F₀

CaCO₃/Pyophage, F₀

Ca₃(PO₄)₂/Pyophage, F₀

CaCO₃/Intestiphage, F₀

The filtrates were analyzed for Plaque Forming Units (PFU) to determinewhich portions of phages were adsorbed by solid adsorbents and whichportion was lost with the filtrates.

The phage-containing (phage-immobilized) wet solid products that wasobtained on the filter contained about 0.7 L (20-23) % of the initialliquid. The obtained wet solid products were moved to sterile glassvessels. These wet (W) solids were referred below as Salt/Bacteriophage,W₀:

MgCO₃/Staphylophage, W₀

CaCO₃/Staphylophage, W₀

Ca₃(PO₄)₂/Staphylophage, W₀ (see FIG. 4 for the activity of this wetsolid)

CaCO₃/Pyophage, W₀

Ca₃(PO₄)₂/Pyophage, W₀

CaCO₃/Intestiphage, W₀ (see FIG. 5 for the activity of this wet solid)

The wet solids obtained were used for:

(i) quantitative analysis (PFU determination after desorption ofphages),

(ii) washing (see Step 3), and

(iii) drying (see Step 4).

Analysis

Analysis Filtrates—Salt/Bacteriophage, F₀: Aliquots was removed from thefiltrates and PFUs were determined using the double agar overlay methodof Gratia (Gratia A. Des relations numériques entre bactéries lysogèneset particules de bactériophage. Annales de l'Institut Pasteur 57:652-676(1936), and Gratia J.-P. Andre Gratia: A forerunner in microbial andviral genetics. Genetics 156:471-476 (2000). The filtrates were alsosubjected to UV-analysis with the purpose to determine which portion ofadmixtures were removed after Steps 1 and 2. Admixtures refer to, forexample, both proteins and products of bacterial lysis—debris of nucleicacids (absorb at 260 nm) and proteins (absorb at 280 nm). See, e.g.F.-X. Schmid, Biological Macromolecules: UV-visible Spectrophotometry.ENCYCLOPEDIA OF LIFE SCIENCES/& 2001 Macmillan Publishers Ltd, NaturePublishing Group/www.els.net). A wide absorption pick in the region250-300 nm (see FIGS. 1 and 2 ) corresponds to a mixture of debris ofnucleic acids and proteins.

Desorption of phages from Salt/Bacteriophage, W₀: 10.0 g of the wetsolid (that corresponds to ca. 3.0 g of the dry adsorbent) and 30.0 mLof saline solution (w/v ratio 1:10 per dry adsorbent—the same as in theStep 1 above) were placed in a 250.0 mL flat-bottom flask, sealed with astopper and shook for 15 min. The flask was removed from a shaker andkept until the solid was precipitated. Right after the solid wasprecipitated, an aliquot was carefully removed from the supernatant forthe determination of PFUs using the double agar overlay method of Gratia(Gratia A. Des relations numériques entre bactéries lysogènes etparticules de bactériophage. Annales de l'Institut Pasteur 57:652-676(1936), and Gratia J.-P. Andre Gratia: A forerunner in microbial andviral genetics. Genetics 156:471-476 (2000)) (see Results sectionbelow).

Step 3. Washing of the Obtained Solid Product by Saline Solution(Optional; can be Done for Several Times in Case of Need)

Salt/Bacteriophage, W₀ product (ca. 1.0 kg—0.3 kg of the adsorbent+0.7kg of adsorbed water) obtained after Step 2 was treated by saline (0.9%NaCl solution) similar to the procedure described in the Step 1: to themoist solid (1.0 kg) was added 3.0 L of saline solution and the mixturewas thoroughly homogenized by stirring for 20-30 min. The stirring wasstopped, the mixture was kept at r.t. for additional 30 min withoutagitation and filtered off.

The filtrates obtained after washing the Salt/Bacteriophage, W₀. werereferred below as Salt/Bacteriophage, F₁:

MgCO₃/Staphylophage, F₁

CaCO₃/Staphylophage, F₁

Ca₃(PO₄)₂/Staphylophage, F₁

CaCO₃/Pyophage, F₁

Ca₃(PO₄)₂/Pyophage, F₁

CaCO₃/Intestiphage, F₁

The filtrates obtained after Step 3 were analyzed for PFU to determinewhich portions of phages were adsorbed by the solid adsorbents and whichportions were lost with the filtrates.

The wet solid products obtained on the filter were moved to sterileglass vessels. These wet solids obtained after washing were referredbelow as

Salt/Bacteriophage, W₁:

MgCO₃/Staphylophage, W₁

CaCO₃/Staphylophage, W₁

Ca₃(PO₄)₂/Staphylophage, W₁

CaCO₃/Pyophage, W₁

Ca₃(PO₄)₂/Pyophage, W₁

CaCO₃/Intestiphage, W₁

The obtained wet solids were used for:

(i) quantitative analysis (PFUs determination after desorption ofphages), and

(ii) drying (see Step 4).

Analysis

Filtrates: Salt/Bacteriophage, F₁: Aliquots was removed from thefiltrates and PFUs were determined using the double agar overlay methodof Gratia. The filtrates were also subjected to UV-analysis with thepurpose to determine which portion of admixtures were removed after theSteps 3 (See Results below).

Desorption of phages from Salt/Bacteriophage, W₁: 10.0 g of the wetsolid (that corresponds to ca. 3.0 g of the dry adsorbent) and 30.0 mLof saline solution (w/v ratio 1:10 per dry adsorbent—the same as in theStep 1 above) were placed in a 250.0 mL flat-bottom flask, sealed with astopper and shook for 15 min. The flask was removed from a shaker andkept until the solid precipitated. Right after the solid wasprecipitated, an aliquot was carefully removed from the supernatant forthe determination of PFUs using the double agar overlay method of Gratia(See Results below).

Step 4. Drying of the Obtained Solid Products Salt/Bacterio-Phage, W₀ orSalt/Bacteriophage, W₁

The wet solids obtained on the filters after the Step 2 or Step 3 weresubjected to drying using 3 different methods:

-   -   Vacuum drying at 40-45° C. over a water adsorbent (anhydrous        CaCl₂ or Na₂SO₄, or silica gel),    -   Freeze-drying, and    -   Spray-drying.

Vacuum drying is the simplest drying method as it does not require theuse of complex and expensive equipment. However, vacuum drying requiresthe use of water adsorbents (anhydrous CaCl₂ or Na₂SO₄, or silica gel)that should be regenerated (dried at 200-250° C. to remove water) beforethe repeated use.

Freeze-drying does not require the use of water adsorbent, butfreeze-driers are generally more expensive than vacuum-driers. It shouldbe noted that freeze-drying of Salt/Bacteriophage, W wet solids takesabout 5-fold less time than freeze-drying of liquid bacteriophages sincefreeze drying of Salt/Bacteriophage, W wet solids utilizes 5-fold lesswater.

Spray-drying requires the most expensive equipment, but it is preferableto vacuum drying and freeze-drying for continuous processes.

For freeze-drying, Salt/Bacteriophage, W wet solids are mixed withsaline solutions (1.0 kg of wet solid+3.0 L of saline solution—w/v ratio1:10 per dry adsorbent) to obtain suspensions that are to be supplied(feed) to spray drier. Mild conditions for spray drying bacteriophagesare found—air temperature at the inlet 90-95° C., and 50° C. at theoutlet; the latter is to be regulated by the selection of suspension'sappropriate feed rate. In this case, dry powdery preparations containNaCl (27 g that comes from 3.0 L of saline solution).

The dried solids to be obtained after drying of Salt/Bacteriophage, W₀are labeled as Salt/Bacterophage, W₀VD (vacuum dried) orSalt/Bacteriophage, W₀FD (Freeze-dried)

Results Before Drying

In the tables given below, the results for cases 1-3 were obtained afterStep 1, Step 2, and Step 3. The results for cases 4-6 were obtainedafter Step 1.

Case 1: MgCO₃ + Staphylophage Sample Liquid bacteriophage # sample PFUpH 1 Staphylococcus aureus * 0.9 × 10⁸ 7.74 2 MgCO₃/Staphylophage,F₀ 2.0× 10⁶ 9.47 3 MgCO₃/Staphylophage,W₀ ** 4.0 × 10⁷ 9.48 4MgCO₃/Staphylophage,F₁ 1.0 × 10² 9.52 5 MgCO₃/Staphylophage,W₁ 0.9 × 10⁷9.42 *** * Serial preparation of Biochimpharm, LLC, Tbilisi, Georgia **Desorbed from MgCO₃/Staphylophage,W₀ *** Desorbed fromMgCO₃/Staphylophage,W₁Degree of the adsorption of Staphylophages after the Step1=(0.9×10⁸−2.0×10⁶)/(0.9×10⁸)×100=97.8%.Degree of the adsorption (retention) of Staphylophages after the Step3=(0.9×10⁸−2.0×10⁶−1.0×10²)/(0.9×10⁸)×100≈97.8%Also see FIGS. 1A-C for UV analysis of Sample #1, Sample #2, and Sample#3 in Case 1, respectively.

Case 2: CaCO₃ + Staphylophage Sample Liquid bacteriophage # sample PFUpH 1 Staphylococcus aureus * 2.0 × 10⁸ 7.76 2 CaCO₃/Staphylophage,F₀ 3.0× 10⁶ 7.39 3 CaCO₃/Staphylophage,W₀ ** 1.0 × 10⁸ 7.59 4CaCO₃/Staphylophage,F₁ 1.0 × 10⁵ 7.31 5 CaCO₃/Staphylophage,W₁ 9.0 × 10⁷7.38 *** * Serial preparation of Biochimpharm, LLC, Tbilisi, Georgia **Desorbed from CaCO₃/Staphylophage,W₀ *** Desorbed fromCaCO₃/Staphylophage,W₁Degree of the adsorption of Staphylophages after Step1=(2.0×10⁸−3.0×10⁶)/(2.0×10⁸)×100=98.5%.Degree of the adsorption (retention) of Staphylophages after Step3=(2.0×10⁶−3.0×10⁶-1.0×10⁵)/(2.0×10⁸)×100≈98.0%.Also see FIGS. 2A-C for UV analysis of Sample #1, Sample #2, and Sample#3 in Case 2, respectively.

Case 3: Ca₃(PO₄)₂ + Staphylophage Sample # Liquid bacteriophage samplePFU pH 1 Staphylococcus aureus * 7.0 × 10⁹ 7.65 2Ca₃(PO₄)₂/Staphylophage,F₀ 7.0 × 10⁷ 7.72 3 Ca₃(PO₄)₂/Staphylophage,W₀4.0 × 10⁹ 7.79 ** 4 Ca₃(PO₄)₂/Staphylophage,F₁ 1.0 × 10⁷ 7.77 5Ca₃(PO₄)₂/Staphylophage,W₁ 2.0 × 10⁹ 7.58 *** * Serial preparation ofBiochimpharm, LLC, Tbilisi, Georgia ** Desorbed fromCa₃(PO₄)₂/Staphylophage,W₀ *** Desorbed from Ca₃(PO₄)₂/Staphylophage,W₁Degree of the adsorption of Staphylophages after Step1=(7.0×10⁹−7.0×10⁷)/(7×10⁹)×100=99.0%Degree of the adsorption (retention) of Staphylophages after Step3=(7.0×10⁹−7.0×10⁷−1.0×10⁷)/(7×10⁹)×100≈99.0%.

Case 4: CaCO₃ + Pyophage Sample # Liquid bacteriophage sample PFU pH 1Pyophage* 7.36 E.coli 7.0 × 10⁹ Proteus 8.0 × 10⁹ Staphylococcus 8.0 ×10⁹ Streptococcus 9.0 × 10⁹ Pseudomonas aeruginosa 5.0 × 10⁹ 2CaCO₃/Pyophage,F₀ 7.41 E.coli 2.0 × 10⁶ Proteus 2.0 × 10⁴ Staphylococcus1.0 × 10⁴ Streptococcus 5.0 × 10³ Pseudomonas aeruginosa 1.0 × 10⁵ 3CaCO₃/Pyophage,W₀** 7.66 E.coli 2.0 × 10⁸ Proteus 1.0 × 10⁸Staphylococcus 1.0 × 10⁷ Streptococcus Pseudomonas 1.0 × 10⁶ aeruginosa1.0 × 10⁷ *Serial preparation of Biochimpharm, LLC, Tbilisi, Georgia**Desorbed from CaCO₃/Pyophage,W₀Degree of the adsorption of the phages at Step 1:E. coli=(7.0×10⁹−2.0×10⁶)/(7.0×10⁹)×100≈100%Proteus=(8.0×10⁹−2.0×10⁶)/(8.0×10⁹)×100≈100%Staphylococcus=(8.0×10⁹−1.0×10⁴)/(8.0×10⁹)×100≈100%Streptococcus=(9.0×10⁹−5.0×10³)/(9.0×10⁹)×100≈100%Pseudomonas aeruginosa=(5.0×10⁹−1.0×10⁵)/(5.0×10⁹)×100≈100%

Case 5: Ca₃(PO₄)₂ + Pyophage Sample # Liquid bacteriophage sample PFU pH1 Pyophage* 7.15 E.coli 5.0 × 10⁹ Proteus 3.0 × 10⁹ Staphylococcus 3.0 ×10⁹ Streptococcus 2.0 × 10⁹ Pseudomonas aeruginosa 9.0 × 10⁸ 2Ca₃(PO₄)₂/Pyophage,F₀ 7.59 E.coli 5.0 × 10⁶ Proteus 9.0 × 10⁷Staphylococcus 2.0 × 10⁷ Streptococcus 3.0 × 10⁶ Pseudomonas aeruginosa1.0 × 10⁶ 3 Ca₃(PO₄)₂/Pyophage,W₀** 7.49 E.coli 4.0 × 10⁸ Proteus 5.0 ×10⁸ Staphylococcus 2.0 × 10⁸ Streptococcus 4.0 × 10⁵ Pseudomonasaeruginosa 3.0 × 10⁷ *Serial preparation of Biochimpharm, LLC, Tbilisi,Georgia **Desorbed from Ca₃(PO₄)₂/Pyophage,W₀Degree of the adsorption of the phages at Step 1:E. coli=(5.0×10⁹−5.0×10⁶)/(5.0×10⁹)×100=99.9%Proteus=(3.0×10⁹−9.0×10⁷)/(3.0×10⁹)×100≈97.0%Staphylococcus=(3.0×10⁹−2.0×10⁷)/(3.0×10⁹)×100≈99.3%Streptococcus=(2.0×10⁹−3.0×10⁶)/(2.0×10⁹)×100≈99.8% Pseudomonasaeruginosa=(9.0×10⁶−1.0×10⁶)/(9.0×10⁸)×100≈99.9%

Case 6: CaCO3 + Intestiphage Sample # Liquid bacteriophage sample PFU pH1 Intestiphage* 7.37 E.coli 6.0 × 10⁹ Proteus 9.0 × 10⁹ Staphylococcus7.0 × 10⁹ Enterococcus 1.0 × 10⁹ Pseudomonas aeruginosa 7.0 × 10⁹Shigella 3.0 × 10⁹ Salmonella 1.0 × 10⁹ 2 CaCO₃/Intestiphage,F₀ 4.0 ×10⁶ 7.40 E.coli 8.0 × 10⁴ Proteus 1.0 × 10⁴ Staphylococcus 7.0 × 10⁷Enterococcus 1.0 × 10⁵ Pseudomonas aeruginosa 2.0 × 10⁵ Salmonella 8.0 ×10⁷ 3 CaCO₃/Intestiphage,W₀** 7.60 E. coli 3.0 × 10^(s) Proteus 2.0 ×10⁸ Staphylococcus 3.0 × 10⁷ Enterococcus 5.0 × 10⁷ Pseudomonasaeruginosa 7.0 × 10⁷ Shigella 2.0 × 10⁶ Salmonella 7.0 × 10⁷ *Serialpreparation of Biochimpharm, LLC, Tbilisi, Georgia **Desorbed fromCaCO₃/Intestiphage,W₀Degree of the adsorption of the phages at Step 1:E. coli=(6.0×10⁹−4.0×10⁶)/(6.0×10⁹)×100≈99.9%Proteus=(9.0×10⁹−8.0×10⁴)/(9.0×10⁹)×100≈100.0%Staphylococcus=(7.0×10⁹−1.0×10⁴)/(7.0×10⁹)×100≈100.0%Enterococcus=(1.0×10⁹−7.0×10⁷)/(1.0×10⁹)×100≈93.0%Pseudomonas aeruginosa=(7.0×10⁹−1.0×10⁵)/(7.0×10⁹)×100≈100.0%Shigella=(3.0×10⁹−2.0×10⁵)/(3.0×10⁹)×100≈100%Salmonella=(1.0×10⁹−8.0×10⁷)/(1.0×10⁹)×100≈92%

The filtrates obtained after Step 2 contained too low quantity (in termsof PFUs) of bacteriophages (Samples #2 of the Cases 1-6) compared toinitial PFUs (Samples #1 of the Cases 1-6). This could be connected witheither inactivation of phages or their adsorption by the used salts.

To verify that this was connected with the phages adsorption rather thaninactivation, desorption of the phages from the wet solidsSalt/Bacteriophage, W₀ (Samples #3 of the Cases 1-6) with salinesolution was carried out. It was found that large quantities of thephages comparable with the initial PFUs were desorbed in all 6 Casesstudied. This strongly evidenced that the water-insoluble salts—MgCO₃,CaCO₃, and Ca₃(PO₄)₂ intensively adsorbed the bacteriophages from thewater solutions, and the degree of the adsorption in all cases was over90% (in some cases almost 100%).

After washing (Step 3) of wet solids Salt/Bacteriophage, W₀ with salinesolution, some portions of bacteriophages were lost as determined bymeasuring PFUs of the filtrates Salt/Bacteriophage, F₁ (shown forStaphylophage, Samples #4 of the Cases 1-3). However, the basic portionsof the phages were retained by the salt adsorbents MgCO₃, CaCO₃, andCa₃(PO₄)₂ as proved by high PFUs of the phages desorbed with salinesolution from wet solids Salt/Bacteriophage, W₁ (Samples #5 of the Cases1-3).

Among the salts examined as adsorbents, CaCO₃ and Ca₃(PO₄)₂ appeared toadsorb phages intensively without influencing the pH of bacteriophagesolutions. MgCO₃ appeared to be a good adsorbent, however, it appearedto increase the solutions' pH (makes them more alkaline), which can harmphages. Therefore, taking at the same time into account the positiverole of Mg++ cations in wound healing (Alimohammad A., Mohammadali M.,Mahmod K., Khadijeh S. A Study of the effect of Magnesium hydroxide onthe wound healing process in rats, Medical Journal of Islamic WorldAcademy of Sciences, 16(4), 165-170 (2007)) along with Ca++ cations(Lansdown A. B., Calcium: a potential central regulator in wound healingin the skin. Wound Repair Regen. 10(5), 271-85 (2002)), MgCO₃ can beadded to the dry powdery phage preparation obtained on the basis ofCa-salts.

Results after Drying

A. Vacuum Drying

Protocol of vacuum drying. The wet solid Salt/Bacteriophage, W₀ (orSalt/Bacteriophage, W₁) was placed in a vacuum drier and dried underreduced pressure at 45° C. over water adsorbent (anhydrous CaCl₂ orNa₂SO₄, or silica gel) up to constant weight.

Case 7: CaCO₃ + Pyophage PFU of the phages desorbed from thevacuum-dried powderypreparation CaCO3/Pyophage, W₀VD (PFU of the phagesdesorbed from Components of serial PFU the wet preparation (desorbedPyophage, from the wet preparation E.coli 7.0 × 10⁹ 9.0 × 10⁷ (2.0 ×10⁸) Proteus 8.0 × 10⁹ 8.0 × 10⁷ (1.0 × 10⁸) Staphylococcus aureus 8.0 ×10⁹ 8.0 × 10⁶ (1.0 × 10⁷) Streptococcus 9.0 × 10⁹ 8.0 × 10⁵ (1.0 × 10⁶)Pseudomonas aeruginosa 5.0 × 10⁹ 4.0 × 10⁷ (7.0 × 10⁷)B. Freeze Drying

Protocol of freeze-drying. The wet solid Salt/Bacteriophage, W₀ (orSalt/Bacterio-phage, W₁) was frozen and placed in a freeze-drier anddried up to constant weight.

Case 8: CaCO₃ + Pyophage PFU of the phages desorbed from thefreeze-dried powdery preparation CaCO₃/Pyophage, W₀FD (PFU of the phagesdesorbed from the wet preparation (desorbed from the Components ofserial PFU wetpreparation Pyophage, CaCO₃/Pyophage,W₀) E.coli 7.0 × 10⁹1.0 × 10⁸ (2.0 × 10⁸) Proteus 8.0 × 10⁹ 1.0 × 10⁸ (1.0 × 10⁸)Staphylococcus aureus 8.0 × 10⁹ 9.0 × 10⁶ (1.0 × 10⁷) Streptococcus 9.0× 10⁹ 9.0 × 10⁵ (1.0 × 10⁶) Pseudomonas 5.0 × 10⁹ 6.0 × 10⁷ (7.0 × 10⁷)aeruginosa

To determine the bacteriophages content in the dried preparations, 3.0 gof a powdery (dry) phage preparation and 30.0 mL of saline solution wasplaced in a 250 mL flat-bottom flask, sealed with a stopper and shookfor 15 min. The flask was removed from a shaker and kept until the solidwas precipitated. Right after the solid was precipitated, an aliquot,containing desorbed phages, was removed from the supernatants for thedetermination of PFU using the double agar overlay method of Gratia.

The results given in Cases 7 and 8 above showed that the PFU of thebacteriophages desorbed from the dried preparations CaCO₃/Pyophage, W₀VDand CaCO₃/Pyophage, W₀FD with saline solution were somewhat lowercompared to the PFU of the bacteriophages desorbed from the wetpreparation CaCO₃/Pyophage, W₀ that were given in parentheses (see alsoSample #3 of the Case 4).

In turn, the PFU of the bacteriophages desorbed from the powderypreparation CaCO₃/Pyophage, W₀FD (Case 8) was a little bit higher thanthe PFU of the bacteriophages desorbed from the powdery preparationCaCO₃/Pyophage, W₀VD (Case 7). The obtained results may be connectedwith either inactivation of bacteriophages during drying (maybe less incase of freeze-drying) or incomplete desorption of bacteriophages underthe used conditions (i.e. desorption with saline solution at roomtemperature)—the latter appeared to be more likely.

C. Spray Drying

Spray drying conditions Spray drier: NIRO MOBILE MINOR™ (Denmark)equipped with a rotary atomizer

Bacteriophages: Serial phages of “Biochimpharm, LLC (Georgia) Staph. a.& E. coli

Fillers: NaHCO₃, MgCO₃, and CaCO₃

Filler's concentration, w/v: 8.0%

Pump: Peristaltic

Feed rate: 1.0 L/h

Temperature: on the inlet −90-95° C., on the outlet −50° C.

Protocol of spray drying of liquid phages: 80.0 g of one of the fillers(NaHCO₃, MgCO₃, or CaCO₃) was added to 1.0 L of liquid bacteriophage(Staph. a. or E. coli, serial liquid phages, without purification) andthoroughly stirred. Sodium bicarbonate (NaHCO₃) formed a homogeneoussolution whereas others (MgCO₃ or CaCO₃), formed suspensions (all arereferred to as bacteriophage/salt mixtures).

In a typical procedure a bacteriophage/salt mixture was supplied to NIROMOBILE MINOR™ spray-drier equipped with a rotary atomizer viaperistaltic pump at a feed rate 1.0 L/h. In case of MgCO₃ or CaCO₃bacteriophage/salt mixtures (suspensions) was permanently stirred toavoid the precipitation of the solid (salts). Under these conditions, atthe inlet air temperature 90-95° C., the temperature on the outlet waskept at 50° C. that was mild for bacteriophage drying that resulted inpowdery preparations. The obtained powdery bacteriophage preparationswere subjected to the analysis on bacteriophage content.

Analysis: to determine the bacteriophages content in powdery (dry)preparations. 4.0 g of a powdery phage preparation and 50.0 mL of salinesolution were placed in a 250.0 mL flat-bottom flask, sealed with astopper and shook for 15 min. The flask was removed from a shaker andkept until the solid (in case of MgCO₃ or CaCO₃; in case of NaHCO₃solution is obtained) precipitated. An aliquot was removed from theMgCO₃ or CaCO₃ supernatants right after the solid precipitated, or fromthe NaHCO₃ solution for the determination of PFU using the double agaroverlay method of Gratia.

Results: the results listed as Case 9, showed that, in terms of PFU ofthe bacteriophages in the dried products, the best filler appeared to beCaCO₃ for which PFU for E. coli was either of the same order as initialPFU (decreased 2.5 times only, Sample #1), or for Staphylococcus aureusdecreased by less than two orders (ca. 33 times, Sample #2), followed byMgCO₃ (PFU decreased by 25 and 60 times, accordingly, Samples ##3 and4), and NaHCO₃ (PFU decreased by 250 and 15,000 times, Samples ##5 and6). The latter appeared to be the worst one among the used fillers.

Case 9: Spray drying of liquid bacteriophages in the presence of variousfiller PFU, after drying ** PFU, initial Filler, w/v PFU, (desorbed PFU,after # Bacteriophage % initial* phages drying 1 E.coli CaCO3, 5.0 × 10⁸2.0 × 10⁸ 2.5 8.0 2 Staphylococcus CaCO3, 3.0 × 10⁸ 9.0 × 10⁶ 33.0aureus 8.0 3 E.coli MgCO3, 5.0 × 10⁸ 2.0 × 10⁷ 25.0 8.0 4 StaphylococcusMgCO3, 3.0 × 10⁸ 5.0 × 10⁶ 60.0 aureus 8.0 5 E.coli NaHCO3, 5.0 × 10⁸2.0 × 10⁶ 250.0 8.0 6 Staphylococcus NaHCO3, 3.0 × 10⁸ 2.0 × 10⁴15,000.0 aureus 8.0 *PFU of liquid bacteriophages subjected to spraydrying ** PFU of the bacteriophages desorbed form the dried preparations

The lower results in case of MgCO₃ and NaHCO₃ could be connected withincreasing the solutions' pH that could cause the bacteriophagesinactivation in certain extent (higher in case of NaHCO₃). In contrastto MgCO₃ and NaHCO₃, CaCO₃ could be neutral and did not influence thesolution's pH. Among the used bacteriophages, E. coli appeared to beless tend to the inactivation under the used conditions than S. aureus,though in case of insoluble salts MgCO₃ and CaCO₃, the lowering of PFUcould also be explained by incomplete desorption of phages.

As a whole, due to the mild drying conditions disclosed, the spraydrying with rotary atomizer resulted in bacteriophages survival ateither the same or even higher level compared to the spray drying withpulse combustion atomizer, which was contrary to the statement given inUS Patent Application Publication Number 2009/0093041 A1, published Apr.9, 2009, that a rotary atomizer kills bacteriophages owing to the actionof sharing stress. The good results obtained with rotary atomizer couldbe connected with the presence of solid particles that could adsorbshock thus protecting bacteriophages from the inactivation. Much betterresults (in terms of PFU) obtained after drying with MgCO₃ as comparedwith NaHCO₃ could speak for this assumption since the solutions'alkalization level in both cases were very close (pH 9.0-9.5).

Protocol of spray drying of wet solids obtained after Steps 2 or 3. Forspray-drying Salt/Bacteriophage, W₀ or Salt/Bacteriophage, W₁, wetsolids should be mixed with saline solutions (1.0 kg of wet solid+3.0 Lof saline solution—w/v ratio 1:10 per dry adsorbent) to obtainsuspensions that could be supplied (feed) to spray drier. The dryingconditions were the same as above (inlet air temperature 90-95° C., thetemperature on the outlet was kept at 50° C.; feed rate 1.0 L/h). Inthis case the obtained dry powdery preparations contained NaCl (27 gthat came from 3.0 L of saline solution).

In one example, a powdery salt with immobilized Pyophage was prepared(1:10 w/v ratio) by adding 10.0 L of liquid bacteriophage with anactivity of 10⁸-10⁹ PFU (plaque forming units) to 1.0 kg of medicalgrade calcium carbonate CaCO₃ with an average particle size of 50 μm atroom temperature. The mixture of calcium carbonate and bacteriophage wasthoroughly homogenized by stirring for 20-30 minutes. After cessation ofstirring, the mixture was incubated at room temperature for 30 minwithout agitation. The suspension was then filtered off to obtainseparated wet solid and filtrate. The wet solid was dried by eithervacuum drying at 40-45° C. over a water adsorbent material (anhydrousCaCl₂ or Na₂SO₄, or silica gel), freeze-drying, or spray drying asdisclosed above.

The resulting dried powder comprised calcium carbonate with immobilizedbacteriophages. A powder of medical grade magnesium carbonate with anaverage particle size of 200 μm was added to the dried powder at aweight ratio of 5/95 (MgCO₃/CaCO₃) and thoroughly homogenized. Theobtained mixture was labeled as (Ca₉₅Mg₅)CO₃/Bacteriophage.

Example 5: Preparation of the Second Composition

To a 8 L solution containing 0.6 kg of the poly (ester urea amide)prepared above was added 0.6 kg of (Ca₉₅Mg₅)CO₃/bacteriophage. Theresulting solution was thoroughly homogenized. To the resultingpolymer/bacteriophage suspension, 0.1 kg of benzocaine (or lidocaine),0.07 kg of ciprofloxacin, and 0.007 kg α-chymotrypsin were added andthoroughly homogenized again.

The obtained mixture was poured onto a hydrophobic surface (Teflon®dishes (25×26 cm×cm)) and the chloroform was completely evaporated atroom temperature under atmospheric pressure. The films were then driedat 40° C. under reduced pressure for 24 hr. The films were removed fromthe hydrophobic surface, perforated, and packed. See FIG. 4 forillustrative purpose of the perforated films.

Alternatively, serial liquid phages may be used for preparing the secondcomposition. A solution of the poly(ester urea amide) prepared asdescribed above and liquid phage are mixed vigorously until theformation of fine emulsion. Different ingredients such as various Ca andMg salts or other salts, as well as various bioactive agents can beadded to this emulsion. The obtained emulsion is then cast onto ahydrophobic surface, quickly frozen, and subjected to freeze-drying. Thefilms are removed from the hydrophobic base, perforated, and packed.

When grindable poly (ester urea), i.e., LMW-1L6, replaced poly (esterurea amide) to prepare the second composition, the film formed after theevaporation of organic solvent (chloroform, dichloromethane) wasbrittle. The film was grinded into a fine powder and sieved through thesieve of the desirable mesh-size that can result in microparticlessuitable for the application in spray wound dressing.

Example 6: Preparation of a Poly(Ester Urea Amide)(1L6)_(0.6)-(8L6)_(0.4)

The homo-poly(ester amide) 8L6 composed of L-leucine (L), 1,6-hexanediol(6) and sebacic acids (8), obtained by interfacial polycondensation (IP)of L6 with sebacoyl chloride, may be used for preparingbacteriophage-containing compositions. However, due to its amorphousnature and low glass transition temperature T_(g) of 37° C. (asdescribed in R. Katsarava, V. Beridze, N. Arabuli, D. Kharadze, C. C.Chu, C. Y. Won. Amino acid based bioanalogous polymers. Synthesis andstudy of regular poly(ester amide)s based on bis(α-amino acid)α,ω-alkylene diesters and aliphatic dicarboxylic acids. J. Polym. Sci.:Part A: Polym. Chem. 37, 391-407 (1999).), 8L6 is too pliable and stickyfor many applications. To reduce pliability and stickiness, 8L6 could bemodified to contain some relatively rigid fragments to increase T_(g).In the present example, more rigid fragments of poly(ester urea),composed of L-leucine, 1,6-hexanediol and carbonic acid, referred toherein as 1L6, were incorporated into the poly(ester amide)'s 8L6backbone. The increase rigidity of the poly(ester urea) fragments may beattributed to their dense intermolecular hydrogen bond networks.

To incorporate poly(ester amide) fragments into the 8L6 backbone, atleast one sebacic acid is replaced by at least one carbonic acid in thepolymeric backbone. Replacing sebacic acid in the polymeric backbonewith carbonic acid may increase the T_(g) up to 60° C.

In one example, due to the distribution of poly(ester amide) andpoly(ester urea) blocks in its backbone, the product poly(ester ureaamide) may be labelled (1L6)_(0.6)-(8L6)_(0.4). The product poly(esterurea amide) is synthesized by interfacial polycondensation ofdi-p-toluenesulfonic acid salts of bis-(L-leucine)-1,6-hexylene diesterwith a mixture of sebacoyl chloride/triphosgene at 60/(40:3) mole/moleratio. A two phase system dichloromethane/water is used to carry out theinterfacial polycondensation, which may be completed in as little as 15to 20 min. Sodium carbonate is also added to the mixture to catch thebyproducts p-toluenesulfonic acid, which comes from amino acid basedmonomer, and hydrogen chloride, which is produced by the interaction ofacid chlorides with primary amino groups. These byproducts are highlywater soluble and are retained in the water phase as well as in theexcess of sodium carbonate. Following the interfacial synthesis, thetarget polymer is retained in the organic (dichloromethane) phase. Afterwashing with water, the target polymer in solution may be be used forpreparing second compositions described herein without separating thetarget polymer. Advantageously, dichloromethane is less toxic that manyother organic solvents commonly used to synthesize polymers similar tothe present example. For example, dichloromethane is considered to beabout 10 times less toxic than chloroform.

The amino acid based monomer used in the synthesis described above(di-p-toluenesulfonic acid salts of bis-(L-leucine)-1,6-hexylene dieste)may be prepared by direct condensation of 2.0 moles of L-leucine with1.0 mole of 1,6-hexanediol in the presence of 2.1 moles ofp-toluenesulfonic acid monohydrate in refluxed cyclohexane. Cyclohexaneis less toxic than benzene and toluene, two organics solvents used inalternative processes for prearing amino acid based monomers. Anothermonomer used in the interfacial synthesis is sebacoyl chloride, areadily purchasable product, which facilitates the preparation of thetarget polymer. In the present example, sebacoyl chloride is useddirectly for synthesizing the target polymer. Moreover, in the presentexample, a part of the sebacoyl chloride is substituted by the lessexpensive triphosgene, making the preparation of the target polymer morecost effective than the synthesis of some alternative amino acid basedpolymers. Moreover, in contrast to some previously prepared amino acidbased polymers, the target polymer does not contain L-phenylalanine. Thepresence of L-phenylalanine in a polymer may led to adverse events inpatients suffering from phenylketonuria.

Example 7: Alternative Preparations of Powdery Bacteriophages (the FirstComposition)

A. Powdery Salts

Powdery salts with immobilized bacteriophages were prepared by theincubation of at least one salt in a liquid preparation of at least onebacteriophage and subsequent drying using vacuum-drying orfreeze-drying.

In one instance, 50.0 g of CaCO₃ is added to a 250 mL Erlenmeyer flaskthough a funnel, followed by the addition of 50 mL of liquidbacteriophage in TMN (Tris-MgCl₂—NaCl) buffer to the flask. A Teflonmagnetic stirring bar (l=5-6 cm) is placed in the flask containing themixture, and the flask is then sealed and its contents stirred using amagnetic stirrer for 1.0 hr.

After 1.0 hr, stirring is stopped, the flask is opened, and the contentsof the flask are transferred to a porous glass filter. The contents arefiltered under reduced pressure, and the resulting wet solid istransferred to a glass vessel using a spatula. The non-sealed glassvessel is placed in a vacuum drier containing a pan with 200 mganhydrous sodium sulfate (CAS number: 7757-82-6). The wet solid is thendried under pressure at 40° C. or less for 5 hours. Following removal ofthe vacuum, the glass vessel is removed from the freeze-drier andsealed. The sealed glass vessel containing the powdery salts withimmobilized bacteriophages is stored at 4° C. until use.

In another instance, 50.0 g of CaCO₃ and 50 mL of liquid bacteriophagein TMN (Tris-MgCl₂—NaCl) buffer are placed in a 250 mL round bottomflask. The flask is placed on a rotary evaporator with a joint of thesame size as the round bottom flask and rotated without vacuum for 1.0hr. A vacuum is then applied to the rotary evaporator, and the contentsof the flask are dried at 40° C. until dry solid is formed based onvisual inspection. Vacuum is removed, and the round bottom flask isremoved from the rotary evaporator. The dried product is moved to asterile vessel, and the mass of the product is determined after sealingof the vessel. The expected mass of the product is 51.3 g.

The sealed vessel containing the powdery salts with immobilizedbacteriophages is stored at 4° C. until use.

Sterile, medical grade CaCO₃ with a mean particle size of 6-8 μm is usedto prepare the powdery salts with immobilized bacteriophages.

B. Freeze-Dried Bacteriophage Compositions

In one instance, 10 mL of liquid bacteriophage (10⁹ to 10¹⁰ PFU/mL) inTMN buffer is freeze-dried to prepare freeze-dried TMN buffer withincrusted (impregnated) bacteriophages.

Before freeze drying the liquid bacteriophage in TMN, the freeze-drieris switched on, all stopcock are closed, and a vacuum is applied. Therubber joint of the freeze-drier is cleaned using a sterilizing liquidsuch as chloroform prior to use.

10 mL of liquid bacteriophage in TMN is transferred to a 100 mL flask.The flask is closed with a stopper and transferred to a cooling bath(−10 to −15° C.). The stopper is loosened on the cooled 100 mL flask,which is placed on the freeze-drier after equilibrating to −10° C. to−15° C. The stopcock on the freeze-drier is then opened, and a vacuum isapplied to the flask. The contents of the flask are lyophilized on thefreeze-drier until a white crystalline powder forms.

After a white crystalline powder is formed, the vacuum is removed, andthe flask is removed from the freeze-drier. The mass of the product isdetermined, and the freeze-dried powdery bacteriophage is stored in asealed container at 4° C.

Powdery bacteriophage without salt may be prepared by dialyzing theliquid bacteriophage in TMN in water prior to cooling and freeze-dryingthe liquid bacteriophage.

C. Alternative Analysis Procedure

To determine the bacteriophages content in powdery (dry) preparations.1.0 g of a powdery phage preparation and 1.0 mL of saline solution areplaced in a test tube, sealed with a cap and shook for 30 min in alaboratory shaker. The test tube was removed from a shaker andcentrifuged at 3000 rpm for 15 min. An aliquot is removed fromsupernatant after centrifugation for the determination of PFU using thedouble agar overlay method of Gratia.

Powdery bacteriophage compositions prepared by the procedures above arenon-immunogenic. In addition, powdery water insoluble carbonate saltswith immobilized bacteriophages may protect the bacteriophages when thefirst composition is used in the treatment of a wound with increasedacidity (acidosis).

Example 8: Preparation of the Second Composition

A 4 mL solution containing 13% w/v of poly (ester urea amide) indichloromethane was poured into a cylindrical sterile glass vesselcontaining 30 to 35 mg of freeze-dried bacteriophage. The poly(esterurea amide) was prepared as in Examples 3 or 6 above, and thefreeze-dried bacteriophages were prepared as described in Example 7. Theresulting solution was thoroughly homogenized at a speed to 100 rpm orlower to avoid splashing.

The obtained mixture was poured onto a hydrophobic surface (a sterileTeflon® dish) and the dichloromethane was evaporated at room temperatureunder atmospheric pressure for 4 days. The films were then dried at 37°C. under reduced pressure until the film reached a constant weight.

Dry films were stored at 4° C. until further use.

Example 9: Preparation of the Biodegradable Poly(Ester Urea)

This example shows how a poly(ester urea) as described above may beprepared. 183.94 g (0.267 mol) of di-p-toluenesulfonic acid salt ofbis(L-leucine)-1,6-hexylene diester, L6 (M=688.91), 26.41 g(0.267/3=0.089 mole) of triphosgene (M=296.748 g/mol) Bis-electrophile,0.267 mol, 84.667 g of anhydrous sodium carbonate (0.8 mol), 0.8 L ofamylene-stabilized dichloromethane, 1.6 L of distilled water and 100 ganhydrous sodium sulfate were measured. The bis-electrophilicmonomer—di-p-toluenesulfonic acid salt of bis(L-leucine)-1,6-hexylenediester, anhydrous sodium carbonate, and distilled water were combinesand stirred until complete dissolution of the ingredients (this takesabout 30-40 min). This creates a first solution. Afterwards, thebis-nucleophilic monomer—triphosgene and 0.8 L of amylene-stabilizeddichloromethane were mixed until a homogeneous solution is formed. Thiscreates a second solution. The second solution is then mixed into thefirst solution and stirred, following which stirring is stop to allow atwo phase system to clearly form. The goal polymer is dissolved DCM,which represents the lower layer. The upper layer is water withdissolved in it carbonate salts and by-products of the polycondensation(i.e. polymer synthesis) such as sodium chloride (NaCl) and sodiump-toluenesulfonate (NaOTos). This upper layer is removed and thrownaway. A first wash is performed by adding to the container containingDCM layer with the dissolved polymer 2.0 L of a fresh portion ofdistilled water. The mixtured is then mixed again, for example using astirrer at 100-200 rpm for 30 min. Once again, the resulting mixture isleft still until a two phase system is clearly formed and the waterphase is removed. Additional similar washes, for example 5 total washes,may also be performed. The DCM layer with dissolved polymer may stillcontains some water, which can be extracted using the anhydrous sodiumsulfate. The resulting DCM solution includes the desired polymer.

Example 10: Preparation of the Biodegradable Poly(Ester Urethane Urea)

This example shows how a poly(ester urethane urea) as described abovemay be prepared. A copolymer—poly(ester urethane urea), PEURU,(D6L6)_(0.20n)-(1L6)_(0.80n), where D6 means the residue of1,6-hexanediol in ester-urethane blocks—comes from 1,6-hexamethylenebis(chloroformate), 1—the residue of carbonic acid in ester-ureablocks—comes from phosgene (triphosgene) is prepared as follows. Thefollowing is prepared: 183.94 g (0.267 mol) of di-p-toluenesulfonic acidsalt of bis(L-leucine)-1,6-hexylene diester, L6 (M=688.91); 12.883 g(0.053 mol) of 1,6-Hexamethylene bis(chloroformate) (M=243.08 g/mol);21.158 g (0.214/3=0.0713 mole) of triphosgene (M=296.748 g/molBis-electrophiles—0.053+0.214=0.267 mol; 84.667 g of anhydrous sodiumcarbonate (0.8 mol); 0.8 L of amylene-stabilized dichloromethane; 1.6 Lof distilled water and 100 g anhydrous sodium sulfate. Then, thebis-electrophilic monomer—di-p-toluenesulfonic acid salt ofbis(L-leucine)-1,6-hexylene diester, anhydrous sodium carbonate, anddistilled water are combined and stirred at room temperature untilcomplete dissolution of the ingredients. This forms a first solution.The bis-nucleophilic monomers—1,6-hexamethylene bis(chloroformate) andtriphosgene are then combined and 0.8 L of amylene-stabilizeddichloromethane is added, further to which stirring is performed until ahomogeneous solution is formed. This forms a second solution. The secondsolution is then added slowly to the first solution while the latter isstirred, following which the resulting mixture is stirred until thepolymerisation reaction is complete, for example an additional 15-20min. Then, the water and DCM phases are left to separate, the waterphase is removed, and multiple washes following by drying are performedas in example 9.

Example 11: Preparation of the Biodegradable Poly(Ester Amide Urethane)

This example shows how a poly(ester amide urethane) as described abovemay be prepared: PEAUR, (8L6)_(0.90n -)(D6L6)_(0.10n), where 8 means theresidue of sebacic acid in ester amide block—comes from sebacoylchlioride, D6—the residue of 1,6-hexanediol in ester-urethaneblocks—comes from 1,6-hexamethylene bis(chloroformate). The following isprepared: 183.94 g (0.267 mol) of di-p-toluenesulfonic acid salt ofbis(L-leucine)-1,6-hexylene diester, L6 (M=688.91); 57.47 g (0.267-0.9)0.2403 mol of sebacoyl chloride (M=239.14 g/mol); 6.49 g(0.267-0.1)=0.0267 mol of 1,6-hexamethylene bis(chloroformate) (M=243.08g/mol) Bis-electrophiles −0.2403+0.0267=0.267 mol; 84.667 g of anhydroussodium carbonate (0.8 mol); 0.8 L of amylene-stabilized dichloromethane;1.6 L of distilled water; 100 g anhydrous sodium sulfate. Thebis-electrophilic monomer—di-p-toluenesulfonic acid salt ofbis(L-leucine)-1,6-hexylene diester, anhydrous sodium carbonate, anddistilled water are combined and stirred at room temperature untilcomplete dissolution of the ingredients. This forms a first solution.Then, the bis-nucleophilic monomers—sebacoyl chloride and1,6-hexamethylene bis(chloroformate) are mixed and 0.8 L ofamylene-stabilized dichloromethane is added and stirring is performedunto a homogeneous solution is formed, further to which stirring isperformed until a homogeneous solution is formed. This forms a secondsolution. The second solution is then added slowly to the first solutionwhile the latter is stirred, following which the resulting mixture isstirred until the polymerisation reaction is complete, for example anadditional 15-20 min. Then, the water and DCM phases are left toseparate, the water phase is removed, and multiple washes following bydrying are performed as in example 9.

Example 12: Preparation of the Biodegradable Poly(Ester Urethane)

This example shows how a poly(ester urethane) as described above may beprepared: poly(ester urethane), PEUR, D6L6, where D6 means the residueof 1,6-hexanediol in ester-urethane blocks—comes from 1,6-hexamethylenebis(chloroformate). The following is prepared: 183.94 g (0.267 mol) ofdi-p-toluenesulfonic acid salt of bis(L-leucine)-1,6-hexylene diester,L6 (M=688.91); 64.9 g (0.267 mol) of 1,6-hexamethylenebis(chloroformate) (M=243.08 g/mol) Bis-electrophile −0.267 mol; 84.667g of anhydrous sodium carbonate (0.8 mol); 0.8 L of amylene-stabilizeddichloromethane; 1.6 L of distilled water 100 g anhydrous sodiumsulfate. The bis-electrophilic monomer—di-p-toluenesulfonic acid salt ofbis(L-leucine)-1,6-hexylene diester, anhydrous sodium carbonate, anddistilled water are combined and stirred until complete dissolution ofthe ingredients. This forms a first solution. Then, the bis-nucleophilicmonomer-1,6-hexamethylene bis(chloroformate) is mixed with 0.8 L ofamylene-stabilized dichloromethane, further to which stirring isperformed until a homogeneous solution is formed. This forms a secondsolution. The second solution is then added slowly to the first solutionwhile the latter is stirred, following which the resulting mixture isstirred until the polymerisation reaction is complete, for example anadditional 15-20 min. Then, the water and DCM phases are left toseparate, the water phase is removed, and multiple washes following bydrying are performed as in example 9. Put magnetic bar (4 cm of length)into the flask and seal the flask with the glass stopper. Put the flaskon a magnetic stirrer at r.t. and stir until a homogeneous solution isformed.

Example 13: Preparation of the Biodegradable Poly(Ester Amide UrethaneUrea)

This example shows how a poly(ester amide urethane urea) as describedabove may be prepared: poly(ester amide urethane urea), PEAURU,(8L6)_(0.50n -)(D6L6)_(0.10n)-(1L6)_(0.40n), where 8 means the residueof sebacic acid in ester amide block—comes from sebacoyl chloride,D6—the residue of 1,6-hexanediol in ester-urethane blocks—comes from1,6-hexamethylene bis(chloroformate), 1—residue of carbonic acid inester-urea blocks—comes from phosgene (triphosgene). The following isprepared: 183.94 g (0.267 mol) of di-p-toluenesulfonic acid salt ofbis(L-leucine)-1,6-hexylene diester, L6 (M=688.91); 31.93 g (0.267-0.5)0.1335 mol of sebacoyl chloride (M=239.14 g/mol); 6.49 g (0.267-0.1)0.0267 mol of 1,6-hexamethylene bis(chloroformate) (M=243.08 g/mol);10.564 g (0.267-0.4)/3=0.1068/3) 0.0356 mol of triphosgene (M=296.748g/mol) Bis-electrophiles−0.1335+0.0267+0.1068=0.267 mol; 84.667 g ofanhydrous sodium carbonate (0.8 mol); 0.8 L of amylene-stabilizeddichloromethane; 1.6 L of distilled water and 100 g anhydrous sodiumsulfate. The bis-electrophilic monomer—di-p-toluenesulfonic acid salt ofbis(L-leucine)-1,6-hexylene diester, anhydrous sodium carbonate, anddistilled water are combined and mixed an triphosgene triphosgene atroom temperature until complete dissolution of the ingredients. Thisforms a first solution. Then, the bis-nucleophilic monomers—sebacoylchloride, 1,6-hexamethylene bis(chloroformate) and triphosgene are mixedand 0.8 L of amylene-stabilized dichloromethane is added, further towhich stirring is performed until a homogeneous solution is formed. Thisforms a second solution. The second solution is then added slowly to thefirst solution while the latter is stirred, following which theresulting mixture is stirred until the polymerisation reaction iscomplete, for example an additional 15-20 min. Then, the water and DCMphases are left to separate, the water phase is removed, and multiplewashes following by drying are performed as in example 9.

Stirring durations are similar in examples 9 to 14 to the examplerelating to PEAU synthesis described above.

Example 14: Alternative Preparation of the Biodegradable Poly(EsterAmide Urea)

This example shows how a poly(ester urethane) as described above may beprepared in a manner that has been optmized with respect to thesynthesis method described above. Di-p-toluenesulfonic acid salt ofbis(L-leucine)-1,6-hexylene diester (2 kg, 2.9 mol) was added to areactor suitable for interfacial polycondensation. Water (9.5 L) wasadded to the reactor with stirring. To the obtained suspension,anhydrous sodium carbonate (0.920 kg, 8.7 mol) was added and stirred atroom temperature for approximately 30-40 min (the first solution). In aseparate vessel, tri-phosgene (0.116 kg, 1.2/3 mol) and sebacoylchloride (i.e., ClCO—(CH)—COCl, 0.400 kg, 1.7 mol) were dissolved in(6.3 L) of amylene stabilized (dichloromethane (DCM)) (the secondsolution). The second solution was rapidly added to the first solutionat room temperature to produce a water/organic mixture. Thewater/organic mixture was stirred vigorously for about 15-20 min.Following cessation of stirring, the mixture was allowed to separatecompletely, resulting in a two-layer system. The lower layer containingpoly(ester urea amide) in dichloromethane was separated and washed 2times (2×5.3 L) with distilled water to remove the salts (sodiumchloride, sodium carbonate/bicarbonate, and sodium p-toluene-sulfonate).To the organic layer, 7 kg of brine and 6 L of dichloromethane wereadded. The dichloromethane layer was separated after each wash step andaqueous top layer was discarded. After 3 hours of the phase separation,the rag layer with aqueous layer was kept and washed with additional 100mL dichloromethane. After washing, the (dichloromethane) layer wasseparated again, dried over anhydrous Na₂SO₄ (2 kg), and filtered off.The solution was poured in a non-stick metal pan under a heat lamp inthe chemical fume hood and was dried until it is no longer tacky(typically 1-2 days). Then, it was dried under vacuum in a Pyrex dishuntil constant weight (30° C.). Putting the sheet of polymer overparchment paper allows it to dry (and soften) while avoiding sticking tothe bottom of the Pyrex dishes. Dichloromethane is considered arelatively safer solvent than chloroform to be used in large scaleproduction of polymers. Moreover, it provides good solubility to theproduct of interest and it is not miscible with water, which facilitiespurification processes. The molar concentration of reagents aredifferent in the two methods. Higher reagent concentration leads to theformation of polymer with higher molecular weight, which was observed inlaboratory scale by viscosity measurement.

This PEAU can also be used, as the above-described one, to preparepatches or films including bacteriophages dispersed therein by simplymixing the bacteriophages (for example liophilized or adsorbed on saltsor other inorganic particles) in the polymer containing DCM, with theaddition of any additional patch or film substance that one may want toinclude, and casting the resulting liquid. In some embodiments, theresulting solution spread on a large surface is left standing until mostof the DCM evaporates, and drying is completed in vacuum.

Films for the Treatment and Prevention of Infection in Wounds

The PEAU polymer, along with the other polymers described in the presentapplication, may be used in a method of treating, reducing or preventingbacterial infection in a wound, the method comprising: applying a filmon the wound, the film including a biodegradable polymer (PEAU or theother polymers described in the present application) with bacteriophagesdispersed therein. PEAU has been found to be particularly advantageousas it has mechanical properties allowing manufacturing relatively thinfilms, for example between 100 μm and 1 mm thick, especially when theprotocol of example 14 is used to manufacture the film. The film isflexible, meaning that it can conform to the shape of the part of thehuman body on which it is applied. The film is typically solid whenapplied, but can be in some embodiments wet.

In some embodiments, the film further includes salt particles dispersedin the polymer, for example CaCO₃ particles. The film may furtherinclude a buffer, for example TNM, to provide a better environment tothe bacteriophages. Additional bactericides, such as, andnon-limitingly, antibiotics silversulfadiazine, silver nitrate ornanocrystalline silver may also be either added to the film, or appliedto the wound prior to application of the claimed film. The proposedtreatment has been observed in vitro to be efficient to reduce bacterialload in antibiotic-resistant bacteria. Also, a synergistic effect hasbeen observed on some bacterial strain between the proposed filmsincluding bacteriophages and silver containing products. Surprisingly,the combination of a silver cream component and the proposed film wasseen in some embodiments to reduce bacterial load with a much largerefficiency than would be presumed from results obtained with either thesilver component or bacteriophages.

The proposed film is relatively thin and designed to be replaced aftersome time if needed. The proposed film is gradually dissolved by woundexudate, based on one or more of the volume of wound exudate producedand the quantity of cell lysis enzymes naturally present in the woundexudate. Adjustment of the film thickness, molecular weight and saltcontent allows adjusting the duration over which the film releases thebacteriophages in the wound. For example, each film is applied for aduration of between 1 and 45 days, and in some embodiments for aduration of between 3 and 7 days. After the film is worn down to asignificant degree or has disappeared altogether, a new film may beapplied to the wound as needed.

Of note, due to its relatively small thickness and to the material used,the film is visually unobtrusive and may even allow visual monitoring ofthe wound without removing the film, which may be useful in a clinicalsetting and improve patient comfort as it eliminates repeated removal ofthe film for inspection. For example, the film is substantiallytransparent when wet so that light diffusion by the film is very smallor non-existent. In a specific example, 95%, 97% or 99% or more of thelight going through the film is neither absorbed, reflected nordiffused, or is other words, transmitted in a straight line, accordingto geometric optics. The patent office image processing system would notallow showing such details in a picture, but films according to theinvention have been produced, which, when applied to the skin, stillallowed to see hair, veins (as appearing trough the skin), pores andother skin surface details almost unaffected by the presence of thefilm, even using magnification. Application on wounds also clearlyallowed to see wound details, such as redness caused by inflammation andwound depth. All these desired optical properties are achievable whilehaving desirable bacteriophage load in the film and suitable mechanicalproperties.

In some embodiments, the bacteriophages are delivered in two phases. Afirst phase, immediately after the film is applied, results in fast (forexample 2 hours or less or 4 hours or less) delivery of a predeterminedfraction of the bacteriophages. Such delivery is either produced byhaving a coating of bacteriophages on the film, or by having a film thatis porous enough to lead to rapid delivery of bacteriophages close tothe surface of the film. This delivery may deliver between 10 and 90percent of the film's bacteriophage load, or even more if bacteriophagesare present on the surface of the film, rapidly and allows to rapidlytreat an already present infection efficiently. Afterwards, theremaining bacteriophages are released at a smaller rate, which helps incompleting elimination of the infection and in preventing furtherinfections. Of note, the film also forms a physical barrier on the skinthat prevents or reduces external contamination. In some embodiments,although the film itself is non-porous, the film is thin enough to allowdiffusion of oxygen therethrough, which further helps in wound healingby reducing the likelihood of infection by anaerobic bacteria.

Gradual liberation of bacteriophages in the second phase is regulated bythe needs of the patient. Indeed, infections typically result in largervolumes of exudates (which dissolves the salts) and the presence ofenzymes (which degrade the matrix), such as elastase andmetalloproteinases, in the exudate. As these components of the exudatereach the film in larger quantities, the film releases larger quantitiesof bacteriophages. To the contrary, if infection is well-controlled, thevolume of exudate is small and the relatively thin film may be preservedintact for a longer duration. In some embodiments, a suitable enzyme,such as the enzymes mentioned in this paragraph, is also added in smallquantity to the film to provide a slow controlled degradation of thefilm even in the absence of infection, to act prophylactically.

The proposed film is typically non-woven and non-porous and includes arelatively large bacteriophage load, for example 500,000PFU/cm{circumflex over ( )}2 or more, which is significant given therelatively small thickness of the film. The proposed film is suitablefor the treatment of any wound, and may also be used in some embodimentsin surgery inside the body. In such applications, the film would be leftinside the body after the surgery is completed to prevent infections.Due to its small size, the film would then disappear relatively quicklyto let the natural healing process occur in the patient. Such anapplication may be useful in contexts in which surgery must be performedin an environment in which sterility is difficult to achieve, such asduring military operations, when far from civilization or even duringspace flight.

The proposed film is also well suited to the treatment of deep wounds,such as pressure ulcers and burn wound, which are traditionallydifficult to treat.

Example 15: Animal Studies Showing Synergy Between Silver Compounds andBacteriophages

Exploratory studies were performed to get indicative data pertaining tothe safety and efficacy of the an SPK cocktail (including bacteriophagesspecific to (P. aeruginosa, S. aureus, and K. pneumoniae) in a murinefull thickness infection model. The first study conducted aimed atassessing the in vivo safety of the phage cocktail and provide anindicative dosage for the mouse injury infection model. The Animal hostis the CD-1 mice, 6-8 week of age, rendered neutropenic bycyclophosphamide. (5 mice per group were studied. The bacterial strainused is Klebsiella pneumonia ATCC 43816. Bacterial challenge was topicalapplication of 1.48×10⁵ CFU/mouse and the test article was a Phageluxphage cocktail SPK, with a total titer of 1.53×10¹⁰ PFU/ml; 1× strengthis defined as 1.00E+08 PFU/mice. Treatment regimen was 10 μL topicaladministration at 0.5, 24, 48, and 72 h post infection. No signs ofanimal distress, body weight changes, or toxicity were found followingrepeated topical applications (SPK control group) or subcutaneousinjections (SPK quality control group) of the SPK cocktail at 0.5, 24,48, and 72 hours. All animals in all control group survived. Nosignificant differences in body weight change were found between the twonon-injured control groups (i.e. Health control and SPK qualitycontrol). No significant differences in wound index were found betweenthe injured control groups (i.e. Injury control and SPK control).Survival curves show that all mice died 2 days post infection for thevehicle control group while SPK cocktail effectively delayed systemicinfection and mice death in a dose-dependent fashion, with higherconcentration of the SPK cocktail being more effective. For the 1×concentration (SPK 10 log PFU/mL), mice death (96 h; p.i) was observedonly following the end of the treatment (72 h).

Survival curves show that treatment with silver sulfadiazine alone couldnot effectively delay systemic infection with all mice dying on day 3post-infection for both the infection control and the silversulfadiazine group. Co-treatment with phage cocktail and silversulfadiazine was superior to silver sulfadiazine standard of care alone,and effectively delayed mice death due to systemic infection.

A second study was carried where the mouse injury model of bacterialinfection was used to evaluate the in vivo efficacy of phage cocktailSPK in preventing mortality for a bis in die dosing regimen. Bacterialchallenge was topical application of 4.83×10⁴ CFU/mouse. Challengeconcentration was reduced in an effort to delay death in the infectiongroup and monitor mice survival for a longer period. The test articlewas Phagelux phage cocktail SPK, of a total titer of 1.53×10¹⁰ PFU/ml;1× strength is defined as 1.00×10⁸ PFU/mice. Treatment regimen was 10 μLtopical administration twice daily for 5 days p.i. Survival curves showthat administration of the SPK cocktail twice daily effectively delayedsystemic infection and rescued mice for a 1E+10 PFU/mL concentration.Only one mice died 3 days post infection for the SPK 1× group whencompared to all 5 mice dying 3 days p.i. for the infection controlgroup. 10¹⁰ PFU/mL was more 10⁹ PFU/mL. Administration of the cocktailtwice daily was more effective than once daily. Mice treated with SPKcocktail were able to recover their body weight when compared to theinfection control group. Bacterial burdens in major organs and woundsites were assessed post mortem for all animals in the infection controland SPK 1× treatment groups. Bacterial burdens were significantly lowerin surviving mice from the group treated with the SPK cocktail whencompared to the Infection control group. SPK cocktail is effective inresolving the systemic bacterial infection.

Phages are self-replicating antibacterial agents, because the phagetiter increases at the infection site until the bacterial host ispresent and efficient concentration may be achieved at exact body/tissuelocation where it is needed. For systemic administrations,bacteriophages are known to be rapidly eliminated from the systemiccirculation by reticuloendothelial system clearance (innate immunemechanisms), then accumulated in spleen and liver or/and inactivated byadaptive immune defense mechanisms involving immunoglobulin in the caseof repetitive application. In our hands, we saw high phageconcentrations at the wound site for all treatments presented, but verylow concentrations of phages in the kidney, spleen, and liver of micethat were either rescued by the phage treatment or for which phagetreatment delayed the systemic infection.

Example 16: In-Vitro Studies Showing Synergy Between Silver Compoundsand Bacteriophages

Silver nanoparticles were extracted from commercially availableSilverlon™ patches. Various bacteria were cultured in cell wells in thepresence of one or more of an antibiotic, silver nanoparticles andbacteriophages, along with control experiments. More specifically,silver nanoparticles were extracted from 10 cm² of Silverlon in 5 mL ofTSB media at 37° C. for 4 h. A. baumannii, K. pneumoniae, P. aeruginosa,S. aureus and B. anthracis specific-strain bacteriophage cocktails wereprepared and the following conditions were investigated in standard 96wells plate (8 replicate for each group): Column 1: 200 μL of TSB(Tryptic soy broth); Column 2: 100 μL of TSB; Column 3: 50 μL of silverextract+50 μL of TSB; Column 4: 50 μL of strain-specific phagecocktail+50 μL of TSB; Column 5: 50 μL of silver extract+50μL ofstrain-specific phage cocktail; Column 6: 50 μL of 2× antibiotic 1 (forwhich bacteria is sensitive)+50 μL of TSB; Column 7: 50 μL of 2×antibiotic 2 (for which bacteria is resistant)+50 μL of TSB. Then, a 1.5mL tube containing 500 μL of TSB+250 μL of phage cocktail+250 μL ofsilver extract was prepared.

Bacteria were cultured to reach OD 0.5 and diluted to OD 0.2. 100 μL ofbacteria suspension was added in columns 2 to 7, and the plates and 1.5mL tube were incubated at 37° C. Absorbance at 600 nm was read at thefollowing timepoints: 0, 1, 2, 3, 4, 5, 6 h. At each timepoint, 100 μLwas removed from the 1.5 mL tube containing silver extract+phagecocktail with titer on corresponding bacteria according to QC-SOP-005.Results are presented on FIGS. 6A to 6D. It can be seen notably that thebacteriophages were superior to the silver nanoparticles, and that forsome bacteria (FIG. 6B), there was a synergy between the bacteriophagesand the silver nanoparticle (Silverlon).

Example 17: In-Vitro Treatment of Wounds: Effect of Film and CeftazidineAntibiotic on K. pneumoniae and on Biofilm Formation

12 mm disks of pig skin were prepared using sterile scissors. Using asterile scalpel, an incision was created in the middle of each pig skindisk and the disks were placed 12 well plates containing TSA (Trypticasesoy agar) (1 mL/well), followed by dispensing 10 μL of bacterial dailyculture (ATCC43816 K. pneumoniae) at 10⁵ cfu/mL. After 10 min, 4 h andovernight static incubation at 37° C., 5 mm*5 mm piece of the proposedfilm were added on the incision zone containing bacteria. After 24 h ofincubation at 37° C., the pig skin was removed from from the TSA layerand fixed using a solution of HEPES+4% PFA+2.5% Glutaraldehyde for 1 hat room temperature, following by washing 3 times with distilled waterand subsequent addition of 30% ethanol and incubation for 30 min at roomtemperature. Afterwards, the sample was washed 3 times with distilledwater. Incubation with ethanol and the associated washes were repeatedwith increases in ethanol concentration (50%, 70%, 90% and 100%). Afterthe last wash, samples were left to dry for 6 h at room temperature andthen placed for several days in the vacuum at room temperature. Sampleswere coated with gold and scanning electron microscope pictures wereobtained. Results are shown in FIGS. 7A to 7F. Superiority of the filmin reducing bacterial contamination was clearly seen by the very smallnumber of bacteria covering the skin surface, when compared to an almostcontinuous carpet of bacteria for control samples and films of bacteriafor the antibiotic group.

Example 18: Production of Thin Films

Thin film mixture comprised of PEAU polymer, bacteriophage cocktail,salts, and organic solvent with a solid content of 19.5% is constantlymixed in a stirred tank reactor connected to an assembly line. The thinfilm blend is metered onto a polymer liner (3M ScotchPak 9741 or otherappropriate lidding material) using a liquid handler/polymer coater.Thickness is for example 0.1 mm to 1 mm, or 0.1 mm to 0.55 mm. This canbe achieved using comma head coating setup. Coating width can be anysuitable width, for example from 80 mm to 550 mm. The lidding film canbe either pre-cut or cut at a later stage in the process. Coating speedcan vary, for example, between 0.25 m/min to 2 m/min. The wet-blendliner is transferred on the conveyer belt into a series of ovens withtemperatures varying from 37.8 to 50° C. in order to evaporate thesolvent without affecting the activity of the bacteriophages. A backinglid is placed, and if not pre-cut, the dry-blend is passed through acutter and a packaging unit.

Example 19: Alternative PEAU Synthesis

In some embodiments, triphosgene is replaced diphenyl carbonate in PEAUsynthesis according to the reaction:

Due to the health hazards of using triphosgene (may cause severe skinburns and eye damage, and acute toxicity if inhaled), it is advantageousto replace triphosgene by diphenyl carbonate (DPC), the reaction mixturewill then be required to be heated at 40° C. for 20 hours in toluene forthe formation of poly ester urea. The change of solvent should notaffect the formation of polyester amide, leading to the ultimateformation of poly ester amide urea (PEAU). DPC is less reactive thantriphosgene, which reacts with the L6 monomer at room temperature.Therefore, dichloromethane (DCM) should be replaced by another solventwith high boiling point. Toluene with b.p of 111° C. or2-methyltetrahydrofuran with b.p. of 80° C. could be used asalternatives.

Also, one may replace sebacoyl chloride with sebacid acid:

If we use Sebacic Acid instead of Sebacoyl Chloride, the reaction wouldrequire much more heat, as this is a dehydration reaction rather than anucleophilic acyl substitution reaction. The reason that heat is neededwhen using Sebacic Acid is because the reaction proceeds with anammonium carboxylate salt intermediate, which requires high temperaturesto remove an H₂O group to form an amide. Another option to synthesizethe polymer is use sebacic acid instead of sebacoyl chloride and heatingthe reaction to 150° C. (using DPC or triphosgene), as this is adehydration reaction rather than a nucleophilic acyl substitutionreaction. The reason that heat is needed when using sebacic acid isbecause the reaction proceeds with an ammonium carboxylate saltintermediate, which requires high temperatures to remove an H₂O to forman amide. In order to avoid heating the reaction at high temperatures,it is to add DCC (N,N′-dicychlohexylcarbodiimide), a coupling agent inthe synthesis to enhance the reaction of sebacic acid and L6 monomer.

The reaction then becomes

Another strategy is to use 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride, usually known as water soluble carbodiimide, (abbreviatedEDCI or EDC), combining with hydroxybenzotriazole (abbreviated HOBt) inone pot synthesis shown in FIG. 2 . The advantages of using EDCI andHOBt as coupling agents include enhancement of amide formation, reactionat room temperature and by products of the coupling reaction are readilysoluble in water, can be easily removed during the purification process.The reaction then becomes

OTHER EMBODIMENTS

The foregoing discussion discloses and describes merely exemplaryembodiments of the disclosure. One skilled in the art will readilyrecognize from such discussion and from the accompanying drawings andclaims, that various changes, modifications and variations can be madetherein without departing from the spirit and scope of the disclosure asdefined in the following embodiments and claims.

1. A polymer selected from

(1) a poly (ester amide urea) wherein at least one diol, at least onediacid, and at least one amino acid are linked together through an esterbond, an amide bond, and a urea bond,

(2) a poly (ester urethane urea) wherein at least one diol and at leastone amino acid are linked together through an ester bond, a urethanebond, and a urea bond,

(3) a poly (ester amide urethane urea) wherein at least one diol, atleast one diacid, and at least one amino acid are linked togetherthrough an ester bond, an amide bond, a urethane bond, and a urea bond,

(4) a poly (ester amide urethane) wherein at least one diol, at leastone diacid, and at least one amino acid are linked together through anester bond, an amide bond, and a urethane bond,

(5) a poly (ester urea) wherein at least one diol and at least one aminoacid are linked together through an ester bond and a urea bond (in otherwords, at least one diol, a carbonic acid, and at least one amino acidare linked together through an ester bond and a urea bond), and

(6) a poly (ester urethane) wherein at least one diol and at least oneamino acid are linked together through an ester bond and a urethanebond,

further wherein

the at least one diol is a compound of formula:

HO—R₁—OH, R₁ is chosen from C₁-C₁₂ alkylene optionally interrupted by atleast one oxygen, C₃-C₈ cycloalkylene, C₃-C₁₀ cycloalkylalkylene,

the at least one diacid is a compound of formula:HO—(CO)—R₃—(CO)—OH, R₃ is C₁-C₁₂ alkylene,

the at least one amino acid is chosen from a naturally occurring aminoacid and non-naturally occurring amino acid.

2. The polymer of embodiment 1, wherein the polymer is selected from

(1) a poly (ester amide urea) wherein at least one diol, at least onediacid, and at least one amino acid are linked together through an esterbond, an amide bond, and a urea bond,

(2) a poly (ester urethane urea) wherein at least one diol and at leastone amino acid are linked together through an ester bond, a urethanebond, and a urea bond,

(3) a poly (ester amide urethane urea) wherein at least one diol, atleast one diacid, and at least one amino acid are linked togetherthrough an ester bond, an amide bond, a urethane bond, and a urea bond,and

(4) a poly (ester amide urethane) wherein at least one diol, at leastone diacid, and at least one amino acid are linked together through anester bond, an amide bond, and a urethane bond,

wherein the at least one diol is a compound of formula:

HO—R₁—OH, R₁ is chosen from C₂-C₁₂ alkylene optionally interrupted by atleast one oxygen, C₃-C₈ cycloalkylene, C₃-C₁₀ cycloalkylalkylene,

the at least one diacid is a compound of formula:HO—(CO)—R₃—(CO)—OH, R₃ is C₂-C₁₂ alkylene,

the at least one amino acid is chosen from a naturally occurring aminoacid and non-naturally occurring amino acid.

3. The polymer of embodiments 1 or 2, wherein the at least one aminoacid is a L- or D-amino acid.

4. The polymer of embodiments 1 or 2, wherein the at least one aminoacid is L-glycine, L-alanine, L-valine, L-leucine, L-isoleucine,L-proline, L-methionine, L-phenylalanine, L-tryptophan, or a D isomerthereof.

5. The polymer of embodiments 1 or 2, wherein the polymer is a poly(ester amide urea) comprising the following two blocks with randomdistribution thereof:

wherein

the ratio of l:m ranges from 0.01:0.99 to 0.99:0.01, l+m=1,

R₁ is chosen from C₁-C₁₂ alkylene optionally interrupted by at least oneoxygen, C₃-C₈ cycloalkylene, C₃-C₁₀ cycloalkylalkylene,

R₃ is C₁-C₁₂ alkylene,

R₂ and R₄ are independently chosen from the side chains of L- andD-amino acids so that the carbon to which R₂ or R₄ is attached has L orD chirality.

6. The polymer of embodiments 1 or 2, wherein the polymer is poly (esterurethane urea) comprising the following two blocks with randomdistribution thereof:

wherein

the ratio of l:m ranges from 0.01:0.99 to 0.99:0.01, l+m=1,

R₁ and R₅ are independently chosen from C₁-C₁₂ alkylene optionallyinterrupted by at least one oxygen, C₃-C₈ cycloalkylene, C₃-C₁₀cycloalkylalkylene,

and

R₂ and R₄ are independently chosen from the side chains of L- andD-amino acids so that the carbon to which R₂ or R₄ is attached has L orD chirality.

7. The polymer of embodiments 1 or 2, wherein the polymer is poly (esteramide urethane urea) comprising the following three blocks with randomdistribution thereof:

wherein

the ratio of l:m:k ranges from 0.05:0.05:0.90 to 0.90:0.05:0.05,l+m+k=1,

R₁ and R₅ are independently chosen from C₁-C₁₂ alkylene optionallyinterrupted by at least one oxygen, C₃-C₈ cycloalkylene, C₃-C₁₀cycloalkylalkylene, and

R₃ is C₁-C₁₂ alkylene, and

R₂ and R₄ are independently chosen from the side chains of L- andD-amino acids so that the carbon to which R₂ or R₄ is attached has L orD chirality.

8. The polymer of embodiments 1 or 2, wherein the polymer is (esteramide urethane) comprising the following two blocks with randomdistribution thereof:

wherein

the ratio of l:m ranges from 0.01:0.99 to 0.99:0.01, l+m=1,

R₁ and R₅ are independently chosen from C₁-C₁₂ alkylene optionallyinterrupted by at least one oxygen, C₃-C₈ cycloalkylene, C₃-C₁₀cycloalkylalkylene,

R₃ is C₁-C₁₂ alkylene, and

R₂ and R₄ are the same and selected from the side chains of L- andD-amino acids so that the carbon to which R₂ or R₄ is attached has L orD chirality.

9. The polymer of any one of embodiments 1-8, wherein R₁ is —(CH₂)₆—.

10. The polymer of any one of embodiments 1, 2, 5, 7, and 8, wherein R₃is —(CH₂)₈—.

11. The polymer of any one of embodiments 5-8, wherein both R₂ and R₄are the side chain of L-leucine.

12. The polymer of embodiment 5, wherein R₁ is —(CH₂)₆—, R₃ is —(CH₂)₈—,and both R₂ and R₄ are the side chain of L-leucine.

13. The polymer of embodiment 12, wherein l is 0.6 and m is 0.4.

14. A polymer blend comprising a first polymer and a second polymer, thefirst polymer being a polymer of any one of embodiments 1-13, and thesecond polymer being selected from a polymer of any one of embodiments1-13 and a poly (ester amide) wherein at least one diol, at least onediacid and at least one amino acid are linked together through an esterbond and an amide bond and wherein the at least one diol, at least onediacid, and at least one amino acid are as defined in embodiment 1, andfurther wherein the first and second polymers are not the same polymer.15. The polymer blend of embodiment 14, wherein the second polymer is apoly (ester amide) in which at least one diol, at least one diacid, andat least one amino acid are as defined in embodiment 1.16. The polymer blend of embodiment 14 or 15, wherein the ratio of thefirst polymer to the second polymer ranges from 0.01:0.99 to 0.99:0.01.17. The polymer blend of embodiment 14 or 15, wherein the ratio of thefirst polymer to the second polymer ranges from 0.05:0.95 to 0.95:0.05.18. The polymer blend of any one of embodiments 14 or 15, wherein theratio of the first polymer to the second polymer ranges from 0.30:0.70to 0.70:0.30.19. The polymer blend of any one of embodiments 14-18, wherein the firstpolymer is a poly (ester urea) and the second polymer is a poly(esteramide).20. The polymer blend of embodiment 19, wherein the poly(ester amide) iscomposed of L-leucine, 1,6-hexanediol, and sebacic acid and thepoly(ester urea) is composed of L-leucine, 1,6-hexanediol, and carbonicacid.21. The polymer blend of embodiment 19, wherein the poly(ester urea)comprises repeating units of:

and the poly(ester amide) comprises repeating units of:

wherein R₁ is chosen from C₁-C₁₂ alkylene optionally interrupted by atleast one oxygen, C₃-C₈ cycloalkylene, C₃-C₁₀ cycloalkylalkylene,

R₃ is C₁-C₁₂ alkylene,

R₂ and R₄ are independently chosen from the side chains of L- andD-amino acids so that the carbon to which R₂ or R₄ is attached has L orD chirality.

22. The polymer blend of any one of embodiments 14-21, wherein the ratioof the first polymer to the second polymer is 0.4:0.6.

23. A composition comprising a polymer of any one of embodiments 1-13 ora polymer blend of any one of embodiments 14-22 and at least onebioactive agent.

24. The composition of embodiment 23, further comprising at least onefiller.

25. The composition of embodiment 24, wherein the at least one fillerincludes at least one of an inorganic salt, sucrose, gelatin, or abuffer.

26. The composition of embodiments 23 or 24, wherein the at least onefiller includes at least one of calcium salts, magnesium salts, ormixtures thereof.

27. The composition of any one of embodiments 23-26, wherein the atleast one filler is a mixture of calcium carbonate and magnesiumcarbonate.

28. The composition of embodiment 24, wherein the at least one fillerincludes TMN buffer.

29. The composition of any one of embodiments 23-28, wherein the atleast one bioactive agent is chosen from an antiseptic, ananti-infective, bacteriophage, a bacteriophage-derived product,endolysins, a phage protein, a phage enzyme, an antibiotic, anantibacterial, an antiprotozoal agent, an antiviral, an analgesic, ananti-inflammatory agent, a steroid, a non-steroidal anti-inflammatoryagent, Prednisolone, Voltaren, a COX-2 inhibitor, an antineoplasticagent, a contraceptive, a central nervous system (CNS) active drug, anhormone, a vaccine, and mixtures thereof.30. The composition of any one of embodiments 23-29, comprising at leastone of calcium gluconate, a phage stabilizing additive, hyaluronidase,fibrinolysin, a fibrinolytic enzyme, methyluracyl, a metabolic processstimulating agent, sodium hydrocarbonate, L-arginine, a vasodilator,mono- and disaccharides, polysaccharides and mucopolysaccharides,metronidazole, an anti-protozoa drug, clotrimazolum, an anti-fungaldrug, thrombin, a hemostatic, a vitamin, or mixtures thereof.31. The composition of any one of embodiments 23-30, wherein the atleast one bioactive agent includes at least one bacteriophage.32. The composition of any one of embodiments 23-31, wherein the atleast one bioactive agent includes a bacteriophage-derived productselected from endolysins, EPS depolymerases, depolymerases, hydrolases,lyases, phage enzymes, phage early proteins, phage holins, and mixturesthereof.33. The composition of any one of embodiments 23-32, wherein the atleast one bioactive agent includes at least one pain reliever.34. The composition of embodiment 33, wherein the at least one painreliever is chosen from benzocaine, lidocaine, tetracaine, pramocaine,dibucaine, and mixtures thereof.35. The composition of any one of embodiments 23-34, wherein the atleast one bioactive agent includes at least one antibiotic.36. The composition of embodiment 35, wherein the at least oneantibiotic is chosen from tetracycline, ciprofloxacin, levofloxacin,mupirocin, neomycin, erythromycin, bacitracin, polymyxin,chlorohexidine, mafenide acetate, silver sulfadiazine, silver nitrate,and mixtures thereof.37. The composition of embodiment 35 or 36, wherein the at least onebioactive agent further includes at least one bacteriophage.38. The composition of embodiment 37, wherein the at least one bioactiveagent further includes at least one pain reliever.39. The composition of any one of embodiments 23-38, wherein the atleast one bioactive agent includes at least one enzyme.40. The composition of embodiment 39, wherein the at least one enzyme ischosen from papain, collagenase, chymotrypsin, trypsin, elastase,fibrinolysin, hyaluronidase, alpha-chymotrypsin, and mixtures thereof.41. The composition of any one of embodiments 23-40, wherein the atleast one bioactive agent includes at least one anti-bacterial agent.42. The composition of any one of embodiments 23-40, wherein the atleast one bioactive agent includes at least one anti-viral agent.43. The composition of embodiment 23, comprising a poly (ester amideurea), at least one bacteriophage, calcium carbonate, magnesiumcarbonate, benzocaine, ciprofloxacin, and chymotrypsin.44. The composition of any one of embodiments 23-43, wherein thecomposition is in the form of a non-woven porous material.45. The composition of embodiment 44, wherein the non-woven porousmaterial is prepared by:

a. mixing a polymer of any one of embodiments 1-13 or a polymer blend ofany one of embodiments 14-22 in a mixture comprising at least one saltand an organic solvent;

b. casting the resulting mixture from step a onto a hydrophobic surface;

c. evaporating the organic solvent to obtain a film; and

d. leaching the at least one salt from the film.

46. The composition of any one of embodiments 23-43, wherein thecomposition is in the form of perforated film.

47. The composition of any one of embodiments 23-43, wherein thecomposition is in the form of a film.

48. The composition of any one of embodiments 23-43, wherein thecomposition is in the form of a spray.

49. The composition of any one of embodiments 23-43, wherein thecomposition is in the form of an unperforated film.

50. The composition of any one of embodiments 23-43, wherein thecomposition is in the form of a gel.

51. The composition of any one of embodiments 23-43, wherein thecomposition is in the form of an hydrogel.

52. The composition of any one of embodiments 23-43, wherein thecomposition is in the form of an ointment.

53. A composition comprising at least one bacteriophage and at least onesalt.

54. The composition of embodiment 53, wherein the at least one salt isinorganic.

55. The composition of embodiment 53 or 54, wherein the at least onesalt is chosen from calcium salts, magnesium salts, strontium salts, andbarium salts.

56. The composition of any one of embodiments 53-55, wherein the atleast one salt is chosen from calcium salts and magnesium salts.

57. The composition of any one of embodiments 53-56, wherein the atleast one salt is chosen from calcium carbonate, calcium phosphate, andmagnesium carbonate.

58. The composition of any one of embodiments 53-57, wherein the atleast one salt is a mixture of calcium carbonate and magnesiumcarbonate.

59. The composition of any one of embodiments 53-58, wherein the atleast one salt is a mixture of calcium carbonate and magnesium carbonateand the weight ratio of MgCO₃ to CaCO₃ is 5:95.

60. The composition of any one of embodiments 53-59, wherein thecomposition is in the form of a dry powder.

61. A composition comprising at least one bacteriophage and at least onebuffer, wherein the composition is in the form of a dry powder.

62. The composition of embodiment 61, wherein the buffer is TMN(Tris-MgCl₂—NaCl) buffer.

63. A wound dressing comprising the composition of any one ofembodiments 23-62.

64. An implantable surgical device comprising the composition of any oneof embodiments 23-62.

65. A food or animal feed additive comprising the composition of any oneof embodiments 23-62.

66. A method for treating agricultural crops, comprising administeringthe composition of any one of embodiments 23-62 on the agriculturalcrops.

67. A method of treating a patient having a wound in need thereofcomprising inserting into the wound or covering the wound with acomposition of any one of embodiments 23-62.

68. The method of embodiment 67, wherein the wound is a superficialwound.

69. The method of embodiment 67, wherein the wound is an ulcerativewound.

70. The method of embodiment 67 or 69, wherein the wound is a deepwound.

71. The method of any one of embodiments 67-70, wherein the wound isopen or infected.

72. The method of any one of embodiments 67-71, wherein the wound istreated prophylactically before any infection is detected.

73. The method of any one of embodiments 67-72, wherein the compositionalso comprises at least one bacteriophage lytic for bacteria found inthe wound.

74. The method of any one of embodiments 67-73, wherein the compositionalso comprises an enzyme capable of hydrolytically cleaving the polymer.

75. A process for preparing the composition of embodiment 61,comprising: mixing at least one bacteriophage and at least one buffer,and drying the mixture through vacuum drying, freeze drying,lyophilization, or spray-drying.

76. The process of embodiment 75, wherein the mixture is dried throughfreeze-drying.

77. A process for preparing the composition of embodiment 53, comprising

mixing and holding (incubating) the at least one salt and at least onebacteriophage,

filtrating the obtained suspension to obtain the at least onebacteriophage adsorbed (immobilized) wet solid product,

washing the obtained wet solid product with saline solution optionally;and

drying the obtained wet solid product through vacuum drying, freezedrying, lyophilization, or spray-drying.

78. A process for preparing the composition of embodiment 31,comprising:

a. mixing the composition of embodiment 53 or 61 with a mixturecomprising an organic solvent and a polymer of any one of embodiments1-13 or a polymer blend of any one of embodiments 14-21; optionallyadding at least one other bioactive agent;

b. casting the resulting mixture from step a onto a hydrophobic surface;and

c. removing the organic solvent to obtain a film.

79. The process of embodiment 78, wherein removing the organic solventincludes evaporating the organic solvent.

80. A process for preparing the composition of embodiment 23,comprising:

a. mixing the at least one bioactive agent with a mixture comprising anorganic solvent and a polymer of any one of embodiments 1-13 or apolymer blend of any one of embodiments 14-21;

b. casting the resulting mixture from step a onto a hydrophobic surface;and

c. removing the organic solvent to obtain a film.

81. A process for preparing the salt of a diester,

comprising:

heating a mixture comprising

HO—R₁—OH, at least one acid that is not an amino acid, and cyclohexane,

wherein

R₁ is chosen from C₁-C₁₂ alkylene optionally interrupted by at least oneoxygen, C₃-C₈ cycloalkylene, C₃-C₁₀ cycloalkylalkylene,

R₂ and R₄ are independently chosen from the side chains of L- andD-amino acids such that the carbon to which R₂ or R₄ is attached has Lor D chirality;

and the at least one acid that is not an amino acid is chosen frominorganic and organic acids such as sulfonic, sulfuric, and hydrochloricacids.

82. The process of embodiment 81, wherein R₂ and R₄ are both the sidechain of L-leucine.

83. The process of embodiment 81 or 82, wherein R₁ is —(CH₂)₆—.

84. The process of any one of embodiments 81-83, wherein the at leastone acid is p-toluenesulfonic acid.

85. A process for preparing the polymer of any one of embodiments 1-13,comprising

a. mixing a salt of the diester and at least one base in water,

b. mixing at least two bis-electrophiles in an organic solvent,

c. mixing the mixtures from step a and b and stirring vigorously,

d. obtaining the organic layer,

wherein the at least two bis-electrophiles is

a mixture of diacid chloride of formula Cl(CO)—R₃—(CO)Cl andtri-phosgene with molar ratio of the diacid chloride:triphosgene rangesfrom 0.95:(0.05/3) to 0.05:(0.95/3) for preparing poly(ester amideurea), or

a mixture of dichloroformate of formula Cl(CO)—O—R₅—O—(CO)Cl andtriphosgene with molar ratio of the dichloroformate:triphosgene rangingfrom 0.95:(0.05/3) to 0.05:(0.95/3) for preparing poly(ester urethaneurea), or

a mixture of diacid chloride of formula Cl(CO)—R₃—(CO)Cl,di-chloroformate of formula Cl(CO)—O—R₅—O—(CO)Cl, and tri-phosgene withmolar ratio of the diacid chloride:dichloroformate:triphosgene rangingfrom 0.90:0.05:(0.05/3) to 0.05:0.05:(0.90/3) for preparing poly(esteramide urethane urea), or

a mixture of diacid chloride of formula Cl(CO)—R₃—(CO)Cl anddi-chloroformate of formula Cl(CO)—O—R₅—O—(CO)Cl with molar ratio of thediacid chloride:di-chloroformate ranging from 0.95:0.05 to 0.05:0.95 forpreparing poly(ester amide urethane), the diester has the followingformula

wherein

R₁ and R₅ are independently chosen from C₁-C₁₂ alkylene optionallyinterrupted by at least one oxygen, C₃-C₈ cycloalkylene, C₃-C₁₀cycloalkylalkylene,

R₃ is C₁-C₁₂ alkylene, and

R₂ and R₄ are the same and selected from the side chains of L- andD-amino acids so that the carbon to which R₂ or R₄ is attached has L orD chirality.

86. A process for preparing a poly (ester amide urea) comprising:

a. mixing triphosgene, diacid HO(CO)—R₃—(CO)OH, and at least one organicbase in an organic solvent, wherein R₃ is C₁-C₁₂ alkylene,

b. mixing a salt of the diester and at least one base in water,

c. mixing the mixtures from step a and b and stirring vigorously, and

d. obtaining an organic layer,

wherein the diester has the following formula:

wherein

R₁ is chosen from C₁-C₁₂ alkylene optionally interrupted by at least oneoxygen, C₃-C₈ cycloalkylene, C₃-C₁₀ cycloalkylalkylene,

R₃ is C₁-C₂ alkylene; and

R₂ and R₄ are independently chosen from the side chains of L- andD-amino acids such that the carbon to which R₂ or R₄ is attached has Lor D chirality.

87. A process for preparing a poly (ester urethane urea) comprising

a. mixing triphosgene, diol HO—R₅—OH, and at least one organic base inan organic solvent,

b. mixing a salt of the diester and at least one base in water,

c. mixing the mixtures from step a and b and stirring vigorously, and

d. obtaining an organic layer,

wherein

the diester has the following formula:

wherein

R₁ and R₅ are independently chosen from C₁-C₁₂ alkylene optionallyinterrupted by at least one oxygen, C₃-C₈ cycloalkylene, C₃-C₁₀cycloalkylalkylene,

and

R₂ and R₄ are independently chosen from the side chains of L- andD-amino acids such that the carbon to which R₂ or R₄ is attached has Lor D chirality.

88. A process for preparing a poly(ester urea) comprising

a. mixing a salt of the diester and at least one base in water,

b. mixing triphosgene in an organic solvent,

c. mixing the mixtures from step a and b and stirring vigorously, and

d. obtaining an organic layer including the poly (ester urea),

wherein

the diester has the following formula:

wherein

R₁ is chosen from C₁-C₁₂ alkylene optionally interrupted by at least oneoxygen, C₃-C₈ cycloalkylene, C₃-C₁₀ cycloalkylalkylene,

and

R₂ and R₄ are independently chosen from the side chains of L- andD-amino acids such that the carbon to which R₂ or R₄ is attached has Lor D chirality.

89. The process of any one of embodiments 86-88, wherein the salt of thediester is a p-toluenesulfonic acid salt of bis-(L-leucine)-1,6-hexylenediester.

90. The process of any one of embodiments 86-89, wherein the at leastone base is an inorganic base.

91. The process of any one of embodiments 86-90, wherein the at leastone base is sodium carbonate.

92. The process of any one of embodiments 86-91, wherein the organicsolvent is chloroform, dichloromethane, or ethyl acetate.

93. The process of any one of embodiments 86-92, wherein the salt of thediester is prepared by the process of embodiment 81.

The invention claimed is:
 1. A method of treating, reducing orpreventing bacterial infection in a wound, the method comprising:applying a flexible film on the wound, the film including abiodegradable polymer with bacteriophages dispersed therein, wherein thepolymer is a poly (ester amide urea) comprising the following two blockswith random distribution thereof:

wherein the ratio of l:m ranges from 0.01:0.99 to 0.99:0.01, l+m=1, R₁is chosen from C₁-C₁₂ alkylene optionally interrupted by at least oneoxygen, C₃-C₈ cycloalkylene, C₃-C₁₀ cycloalkylalkylene,

R₃ is C₁-C₁₂ alkylene, R₂ and R₄ are independently chosen from the sidechains of L- and D-amino acids so that the carbon to which R₂ or R₄ isattached has L or D chirality; further comprising gradually deliveringat least part of the bacteriophages in the wound at a controlleddelivery rate, the controlled delivery rate being controlled by a volumeand composition of a wound exudate produced by the wound while the filmis applied.
 2. The method of claim 1, wherein R₁ is —(CH₂)₆—, R₃ is—(CH₂)₈—, and both R₂ and R₄ are the side chain of L-leucine.
 3. Themethod as defined in claim 1, wherein the film further includes saltparticles dispersed in the polymer.
 4. The method as defined in claim 3,wherein at least part of the bacteriophages is adsorbed on the saltparticles.
 5. The method as defined in claim 4, wherein the saltparticles include CaCO₃ particles.
 6. The method as defined in claim 1,wherein the film further includes a buffer.
 7. The method as defined inclaim 6, wherein the buffer is TMN (Tris-MgCl₂—NaCl) buffer.
 8. Themethod as defined in claim 1, wherein the film further includes at leastone of silversulfadiazine, silver nitrate and nanocrystalline silver. 9.The method as defined in claim 1, wherein the film further includes anantibiotic.
 10. The method as defined in claim 1, where the film isapplied for a duration of between 1 day and 20 days.
 11. The method asdefined in claim 10, wherein the film is applied for a duration ofbetween 3 and 7 days.
 12. The method as defined in claim 10, wherein thefilm is a first film, the method further comprising removing the firstfilm after the duration and applying a second film including thebiodegradable polymer with the bacteriophages dispersed therein on thewound.
 13. The method as defined in claim 1, further comprisingdelivering a fraction of the bacteriophages in the wound within a rapiddelivery period immediately after applying the film, the rapid deliveryperiod being smaller than 4 hours, and afterwards releasing at leastpart of the remaining bacteriophages in the wound at a rate smaller thanwithin the rapid delivery period.
 14. The method as defined in claim 1,wherein the controlled delivery rate increases for an increase involumic production rate of the wound exudate.
 15. The method as definedin claim 1, wherein the controlled delivery rate increases with anincrease in elastase and metalloproteinases quantity present in thewound exudate as a result of cell lysis within the wound.
 16. The methodas defined in claim 1, wherein the film includes an enzyme operative todegrade the film.
 17. The method as defined in claim 16, wherein theenzyme is selected from elastase and a metalloproteinase.
 18. The methodas defined in claim 1, wherein the bacteriophages are present in thefilm at 500,000 PFU/cm² or more.
 19. The method as defined in claim 1,wherein the film is non-woven and non-porous.
 20. The method as definedin claim 1, wherein the film is between 100 μm and 1000 μm thick. 21.The method as defined in claim 1, wherein the wound is a pressure ulcer.22. The method as defined in claim 1, wherein the wound is a burn wound.23. The method as defined in claim 1, wherein a bactericide is appliedto the wound before the film is applied.
 24. The method as defined inclaim 23, wherein the bactericide includes at least one of anantibiotic, silversulfadiazine, silver nitrate and nanocrystallinesilver.
 25. The method as defined in claim 1, wherein the wound containsat least one of antibiotic-resistant and silver-resistant bacteria, thebacteriophage being specific to the at least one of antibiotic-resistantand silver-resistant bacteria.
 26. The method as defined in claim 1,wherein the film is substantially transparent when wet.
 27. The methodas defined in claim 1, wherein the film is a dry solid film.
 28. Themethod as defined in claim 1, wherein the method is for preventingbacterial infection in the wound.
 29. The method as defined in claim 1,wherein the method is for treating bacterial infection in the wound. 30.A method of treating, reducing or preventing bacterial infection in awound, the method comprising: applying a flexible film on the wound, thefilm including bacteriophages dispersed in a biodegradable polymer,wherein the polymer is selected from (1) a poly (ester amide urea)wherein at least one diol, at least one diacid, and at least one aminoacid are linked together through an ester bond, an amide bond, and aurea bond, (2) a poly (ester urethane urea) wherein at least one dioland at least one amino acid are linked together through an ester bond, aurethane bond, and a urea bond, (3) a poly (ester amide urethane urea)wherein at least one diol, at least one diacid, and at least one aminoacid are linked together through an ester bond, an amide bond, aurethane bond, and a urea bond, (4) a poly (ester amide urethane)wherein at least one diol, at least one diacid, and at least one aminoacid are linked together through an ester bond, an amide bond, and aurethane bond, (5) a poly (ester urea) wherein at least one diol and atleast one amino acid are linked together through an ester bond and aurea bond, and (6) a poly (ester urethane) wherein at least one diol andat least one amino acid are linked together through an ester bond and aurethane bond, further wherein the at least one diol is a compound offormula: HO—R₁—OH, R₁ is chosen from C₁-C₁₂ alkylene optionallyinterrupted by at least one oxygen, C₃-C₈ cycloalkylene, C₃-C₁₀cycloalkylalkylene,

the at least one diacid is a compound of formula:HO—(CO)—R₃—(CO)—OH, R₃ is C₁-C₁₂ alkylene, the at least one amino acidis chosen from a naturally occurring amino acid and non-naturallyoccurring amino acid further comprising gradually delivering at leastpart of the bacteriophages in the wound at a controlled delivery rate,the controlled delivery rate being controlled by a volume andcomposition of a wound exudate produced by the wound while the film isapplied.